Variant nucleic acid libraries for glp1 receptor

ABSTRACT

Provided herein are methods and compositions relating to glucagon-like peptide-1 receptor (GLP1R) libraries having nucleic acids encoding for a scaffold comprising a GLP1R binding domain. Libraries described herein include variegated libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/810,377 filed on Feb. 26, 2019; U.S. ProvisionalPatent Application No. 62/830,316 filed on Apr. 5, 2019; U.S.Provisional Patent Application No. 62/855,836 filed on May 31, 2019;U.S. Provisional Patent Application No. 62/904,563 filed on Sep. 23,2019; U.S. Provisional Patent Application No. 62/945,049 filed on Dec.6, 2019; and U.S. Provisional Patent Application No. 62/961,104 filed onJan. 14, 2020, each of which is incorporated by reference in itsentirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 31, 2020, isnamed 44854-787_201_SL.txt and is 1,080,872 bytes in size.

BACKGROUND

G protein-coupled receptors (GPCRs) are implicated in a wide variety ofdiseases. Raising antibodies to GPCRs has been difficult due to problemsin obtaining suitable antigen because GPCRs are often expressed at lowlevels in cells and are very unstable when purified. Thus, there is aneed for improved agents for therapeutic intervention which targetGPCRs.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are antibodies or antibody fragments thereof that bindsGLP1R, comprising an immunoglobulin heavy chain and an immunoglobulinlight chain: (a) wherein the immunoglobulin heavy chain comprises anamino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identicalto that set forth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308,2309, 2317, 2318, 2319, 2320, or 2321; and (b) wherein theimmunoglobulin light chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316. Further provided hereinare antibodies or antibody fragments thereof that binds GLP1R, whereinthe immunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2303; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2310. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2304; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2311. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2305; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2312. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2306; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2313. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2307; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2314. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2308; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2315. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2309, 2317, 2318, 2319; and wherein the immunoglobulin light chaincomprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or100% identical to that set forth in SEQ ID NO: 2316. Further providedherein are antibodies or antibody fragments thereof that binds GLP1R,wherein the antibody is a monoclonal antibody, a polyclonal antibody, abi-specific antibody, a multispecific antibody, a grafted antibody, ahuman antibody, a humanized antibody, a synthetic antibody, a chimericantibody, a camelized antibody, a single-chain Fvs (scFv), a singlechain antibody, a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fvfragment, a single-domain antibody, an isolated complementaritydetermining region (CDR), a diabody, a fragment comprised of only asingle monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof. Further provided herein are antibodies or antibodyfragments thereof that binds GLP1R, wherein the antibody or antibodyfragment thereof is chimeric or humanized. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theantibody has an EC50 less than about 25 nanomolar in a cAMP assay.Further provided herein are antibodies or antibody fragments thereofthat binds GLP1R, wherein the antibody has an EC50 less than about 20nanomolar in a cAMP assay. Further provided herein are antibodies orantibody fragments thereof that binds GLP1R, wherein the antibody has anEC50 less than about 10 nanomolar in a cAMP assay. Further providedherein are antibodies or antibody fragments thereof that binds GLP1R,wherein the antibody is an agonist of GLP1R. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theantibody is an antagonist of GLP1R. Further provided herein areantibodies or antibody fragments thereof that binds GLP1R, wherein theantibody is an allosteric modulator of GLP1R. Further provided hereinare antibodies or antibody fragments thereof that binds GLP1R, whereinthe allosteric modulator of GLP1R is a negative allosteric modulator.Further provided herein are antibodies or antibody fragments thereofthat binds GLP1R, wherein the antibody or antibody fragment comprises aCDR-H3 comprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281,2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296,2297, 2298, 2299, 2300, 2301, or 2302.

Provided herein are nucleic acid libraries comprising a plurality ofnucleic acids, wherein each nucleic acid encodes for a sequence thatwhen translated encodes for an immunoglobulin scaffold, wherein theimmunoglobulin scaffold comprises a CDR-H3 loop that comprises a GLP1Rbinding domain, and wherein each nucleic acid comprises a sequenceencoding for a sequence variant of the GLP1R binding domain. Furtherprovided herein are nucleic acid libraries, wherein a length of theCDR-H3 loop is about 20 to about 80 amino acids. Further provided hereinare nucleic acid libraries, wherein a length of the CDR-H3 loop is about80 to about 230 base pairs. Further provided herein are nucleic acidlibraries, wherein the immunoglobulin scaffold further comprises one ormore domains selected from variable domain, light chain (VL), variabledomain, heavy chain (VH), constant domain, light chain (CL), andconstant domain, heavy chain (CH). Further provided herein are nucleicacid libraries, wherein the VH domain is IGHV1-18, IGHV1-69, IGHV1-8IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, orIGHV4-59/61. Further provided herein are nucleic acid libraries, whereinthe VH domain is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7,IGHV1, or IGHV1-8. Further provided herein are nucleic acid libraries,wherein the VH domain is IGHV1-69 and IGHV3-30. Further provided hereinare nucleic acid libraries, wherein the VL domain is IGKV1-39, IGKV1-9,IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14,IGLV1-40, or IGLV3-1. Further provided herein are nucleic acidlibraries, wherein a length of the VH domain is about 90 to about 100amino acids. Further provided herein are nucleic acid libraries, whereina length of the VL domain is about 90 to about 120 amino acids. Furtherprovided herein are nucleic acid libraries, wherein a length of the VHdomain is about 280 to about 300 base pairs. Further provided herein arenucleic acid libraries, wherein a length of the VL domain is about 300to about 350 base pairs. Further provided herein are nucleic acidlibraries, wherein the library comprises at least 10⁵ non-identicalnucleic acids. Further provided herein are nucleic acid libraries,wherein the immunoglobulin scaffold comprises a single immunoglobulindomain. Further provided herein are nucleic acid libraries, wherein theimmunoglobulin scaffold comprises a peptide of at most 100 amino acids.

Provided herein are protein libraries comprising a plurality ofproteins, wherein each of the proteins of the plurality of proteinscomprise an immunoglobulin scaffold, wherein the immunoglobulin scaffoldcomprises a CDR-H3 loop that comprises a sequence variant of a GLP1Rbinding domain. Further provided herein are protein libraries, wherein alength of the CDR-H3 loop is about 20 to about 80 amino acids. Furtherprovided herein are protein libraries, wherein the immunoglobulinscaffold further comprises one or more domains selected from variabledomain, light chain (VL), variable domain, heavy chain (VH), constantdomain, light chain (CL), and constant domain, heavy chain (CH). Furtherprovided herein are protein libraries, wherein the VH domain isIGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28,IGHV3-74, IGHV4-39, or IGHV4-59/61. Further provided herein are proteinlibraries, wherein the VH domain is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3,IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. Further provided herein areprotein libraries, wherein the VH domain is IGHV1-69 and IGHV3-30.Further provided herein are protein libraries, wherein the VL domain isIGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1,IGLV1-51, IGLV2-14, IGLV1-40, or IGLV3-1. Further provided herein areprotein libraries, wherein a length of the VH domain is about 90 toabout 100 amino acids. Further provided herein are protein libraries,wherein a length of the VL domain is about 90 to about 120 amino acids.Further provided herein are protein libraries, wherein the plurality ofproteins are used to generate a peptidomimetic library. Further providedherein are protein libraries, wherein the protein library comprisesantibodies.

Provided herein are protein libraries comprising a plurality ofproteins, wherein the plurality of proteins comprises sequence encodingfor different GPCR binding domains, and wherein the length of each GPCRbinding domain is about 20 to about 80 amino acids. Further providedherein are protein libraries, wherein the protein library comprisespeptides. Further provided herein are protein libraries, wherein theprotein library comprises immunoglobulins. Further provided herein areprotein libraries, wherein the protein library comprises antibodies.Further provided herein are protein libraries, wherein the plurality ofproteins is used to generate a peptidomimetic library.

Provided herein are vector libraries comprising a nucleic acid libraryas described herein.

Provided herein are cell libraries comprising a nucleic acid library asdescribed herein.

Provided herein are cell libraries comprising a protein library asdescribed herein.

Provided herein are antibodies, wherein the antibody comprises a CDR-H3comprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282,2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297,2298, 2299, 2300, 2301, or 2302.

Provided herein are antibodies, wherein the antibody comprises a CDR-H3comprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282,2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297,2298, 2299, 2300, 2301, or 2302; and wherein the antibody is amonoclonal antibody, a polyclonal antibody, a bi-specific antibody, amultispecific antibody, a grafted antibody, a human antibody, ahumanized antibody, a synthetic antibody, a chimeric antibody, acamelized antibody, a single-chain Fvs (scFv), a single chain antibody,a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fv fragment, asingle-domain antibody, an isolated complementarity determining region(CDR), a diabody, a fragment comprised of only a single monomericvariable domain, disulfide-linked Fvs (sdFv), an intrabody, ananti-idiotypic (anti-Id) antibody, or ab antigen-binding fragmentsthereof.

Provided herein are methods of inhibiting GLP1R activity, comprisingadministering an antibody or antibody fragment as described herein.Further provided herein are methods of inhibiting GLP1R activity,wherein the antibody or antibody fragment is an allosteric modulator.Further provided herein are methods of inhibiting GLP1R activity,wherein the antibody or antibody fragment is a negative allostericmodulator. Further provided herein are methods of treatment of ametabolic disorder, comprising administering to a subject in needthereof an antibody or antibody fragment as described herein. Furtherprovided herein are methods of treatment of a metabolic disorder,wherein the metabolic disorder is Type II diabetes or obesity.

Provided herein are nucleic acid libraries, comprising: a plurality ofnucleic acids, wherein each of the nucleic acids encodes for a sequencethat when translated encodes for a GLP1R binding immunoglobulin, whereinthe GLP1R binding immunoglobulin comprises a variant of a GLP1R bindingdomain, wherein the GLP1R binding domain is a ligand for the GLP1R, andwherein the nucleic acid library comprises at least 10,000 variantimmunoglobulin heavy chains and at least 10,000 variant immunoglobulinlight chains. Further provided herein are nucleic acid libraries,wherein the nucleic acid library comprises at least 50,000 variantimmunoglobulin heavy chains and at least 50,000 variant immunoglobulinlight chains. Further provided herein are nucleic acid libraries,wherein the nucleic acid library comprises at least 100,000 variantimmunoglobulin heavy chains and at least 100,000 variant immunoglobulinlight chains. Further provided herein are nucleic acid libraries,wherein the nucleic acid library comprises at least 10⁵ non-identicalnucleic acids. Further provided herein are nucleic acid libraries,wherein a length of the immunoglobulin heavy chain when translated isabout 90 to about 100 amino acids. Further provided herein are nucleicacid libraries, wherein a length of the immunoglobulin heavy chain whentranslated is about 100 to about 400 amino acids. Further providedherein are nucleic acid libraries, wherein the variant immunoglobulinheavy chain when translated comprises at least 80% sequence identity toSEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319,2320, or 2321. Further provided herein are nucleic acid libraries,wherein the variant immunoglobulin light chain when translated comprisesat least 80% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313,2314, 2315, or 2316.

Provided herein are nucleic acid libraries comprising: a plurality ofnucleic acids, wherein each of the nucleic acids encodes for a sequencethat when translated encodes for a GLP1R single domain antibody, whereineach sequence of the plurality of sequences comprises a variant sequenceencoding for at least one of a CDR1, CDR2, and CDR3 on a heavy chain;wherein the library comprises at least 30,000 variant sequences; andwherein the antibody or antibody fragments bind to its antigen with aK_(D) of less than 100 nM. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 50,000variant immunoglobulin heavy chains and at least 50,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 100,000variant immunoglobulin heavy chains and at least 100,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 10⁵non-identical nucleic acids. Further provided herein are nucleic acidlibraries, wherein a length of the immunoglobulin heavy chain whentranslated is about 90 to about 100 amino acids. Further provided hereinare nucleic acid libraries, wherein a length of the immunoglobulin heavychain when translated is about 100 to about 400 amino acids. Furtherprovided herein are nucleic acid libraries, wherein the variantimmunoglobulin heavy chain when translated comprises at least 80%sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308,2309, 2317, 2318, 2319, 2320, or 2321. Further provided herein arenucleic acid libraries, wherein the variant immunoglobulin light chainwhen translated comprises at least 80% sequence identity to SEQ ID NO:2310, 2311, 2312, 2313, 2314, 2315, or 2316.

Provided herein antagonists of GLP1R comprising SEQ ID NO: 2279 or 2320.Further provided herein are antagonists, wherein the antagonistcomprises an EC50 of no more than 1.5 nM. Further provided herein areantagonists, wherein the antagonist comprises an EC50 of no more than1.0 nM. Further provided herein are antagonists, wherein the antagonistcomprises an EC50 of no more than 0.5 nM. Further provided herein areantagonists, wherein the antagonist is an antibody or antibody fragmentthereof.

Provided herein are nucleic acid libraries, comprising: a plurality ofnucleic acids, wherein each of the nucleic acids encodes for a sequencethat when translated encodes for a GLP1R binding immunoglobulin, whereinthe GLP1R binding immunoglobulin comprises a variant of a GLP1R bindingdomain, wherein the GLP1R binding domain is a ligand for the GLP1R, andwherein the nucleic acid library comprises at least 10,000 variantimmunoglobulin heavy chains and at least 10,000 variant immunoglobulinlight chains. Further provided herein are nucleic acid libraries,wherein the nucleic acid library comprises at least 50,000 variantimmunoglobulin heavy chains and at least 50,000 variant immunoglobulinlight chains. Further provided herein are nucleic acid libraries,wherein the nucleic acid library comprises at least 100,000 variantimmunoglobulin heavy chains and at least 100,000 variant immunoglobulinlight chains. Further provided herein are nucleic acid libraries,wherein the nucleic acid library comprises at least 10⁵ non-identicalnucleic acids. Further provided herein are nucleic acid libraries,wherein a length of the immunoglobulin heavy chain when translated isabout 90 to about 100 amino acids. Further provided herein are nucleicacid libraries, wherein a length of the immunoglobulin heavy chain whentranslated is about 100 to about 400 amino acids. Further providedherein are nucleic acid libraries, wherein the variant immunoglobulinheavy chain when translated comprises at least 90% sequence identity toSEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319,2320, or 2321. Further provided herein are nucleic acid libraries,wherein the variant immunoglobulin light chain when translated comprisesat least 90% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313,2314, 2315, or 2316.

Provided herein are nucleic acid libraries comprising: a plurality ofnucleic acids, wherein each of the nucleic acids encodes for a sequencethat when translated encodes for a GLP1R single domain antibody, whereineach sequence of the plurality of sequences comprises a variant sequenceencoding for at least one of a CDR1, CDR2, and CDR3 on a heavy chain;wherein the library comprises at least 30,000 variant sequences; andwherein the antibody or antibody fragments bind to its antigen with aK_(D) of less than 100 nM. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 50,000variant immunoglobulin heavy chains and at least 50,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 100,000variant immunoglobulin heavy chains and at least 100,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 10⁵non-identical nucleic acids. Further provided herein are nucleic acidlibraries, wherein a length of the immunoglobulin heavy chain whentranslated is about 90 to about 100 amino acids. Further provided hereinare nucleic acid libraries, wherein a length of the immunoglobulin heavychain when translated is about 100 to about 400 amino acids. Furtherprovided herein are nucleic acid libraries, wherein the variantimmunoglobulin heavy chain when translated comprises at least 90%sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308,2309, 2317, 2318, 2319, 2320, or 2321. Further provided herein arenucleic acid libraries, wherein the variant immunoglobulin light chainwhen translated comprises at least 90% sequence identity to SEQ ID NO:2310, 2311, 2312, 2313, 2314, 2315, or 2316.

Provided herein are antibodies or antibody fragments that binds GLP1R,comprising an immunoglobulin heavy chain and an immunoglobulin lightchain: (a) wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or2321; and (b) wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316. Further provided hereinare antibodies or antibody fragments, wherein the immunoglobulin heavychain comprises an amino acid sequence at least about 90% identical tothat set forth in SEQ ID NO: 2303; and wherein the immunoglobulin lightchain comprises an amino acid sequence at least about 90% identical tothat set forth in SEQ ID NO: 2310. Further provided herein areantibodies or antibody fragments, wherein the immunoglobulin heavy chaincomprises an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NO: 2304; and wherein the immunoglobulin light chaincomprises an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NO: 2311. Further provided herein are antibodies orantibody fragments, wherein the immunoglobulin heavy chain comprises anamino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 2305; and wherein the immunoglobulin light chain comprises anamino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 2312. Further provided herein are antibodies or antibodyfragments, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2306; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2313. Further provided herein are antibodies or antibody fragments,wherein the immunoglobulin heavy chain comprises an amino acid sequenceat least about 90% identical to that set forth in SEQ ID NO: 2307; andwherein the immunoglobulin light chain comprises an amino acid sequenceat least about 90% identical to that set forth in SEQ ID NO: 2314.Further provided herein are antibodies or antibody fragments, whereinthe immunoglobulin heavy chain comprises an amino acid sequence at leastabout 90% identical to that set forth in SEQ ID NO: 2308; and whereinthe immunoglobulin light chain comprises an amino acid sequence at leastabout 90% identical to that set forth in SEQ ID NO: 2315. Furtherprovided herein are antibodies or antibody fragments, wherein theimmunoglobulin heavy chain comprises an amino acid sequence at leastabout 90% identical to that set forth in SEQ ID NO: 2309; and whereinthe immunoglobulin light chain comprises an amino acid sequence at leastabout 90% identical to that set forth in SEQ ID NO: 2316. Furtherprovided herein are antibodies or antibody fragments, wherein theantibody is a monoclonal antibody, a polyclonal antibody, a bi-specificantibody, a multispecific antibody, a grafted antibody, a humanantibody, a humanized antibody, a synthetic antibody, a chimericantibody, a camelized antibody, a single-chain Fvs (scFv), a singlechain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fvfragment, a single-domain antibody, an isolated complementaritydetermining region (CDR), a diabody, a fragment comprised of only asingle monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof. Further provided herein are antibodies or antibodyfragments, wherein the antibody or antibody fragment thereof is chimericor humanized. Further provided herein are antibodies or antibodyfragments, wherein the antibody has an EC50 less than about 25 nanomolarin a cAMP assay. Further provided herein are antibodies or antibodyfragments, wherein the antibody has an EC50 less than about 20 nanomolarin a cAMP assay. Further provided herein are antibodies or antibodyfragments, wherein the antibody has an EC50 less than about 10 nanomolarin a cAMP assay. Further provided herein are antibodies or antibodyfragments, wherein the antibody is an agonist of GLP1R. Further providedherein are antibodies or antibody fragments, wherein the antibody is anantagonist of GLP1R. Further provided herein are antibodies or antibodyfragments, wherein the antibody is an allosteric modulator of GLP1R.Further provided herein are antibodies or antibody fragments, whereinthe allosteric modulator of GLP1R is a negative allosteric modulator.

Provided herein are antibodies or antibody fragments, wherein theantibody or antibody fragment comprises a sequence of any one of SEQ IDNOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291,2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302 or asequence set forth in Table 27.

Provided herein are antibodies or antibody fragments, wherein theantibody or antibody fragment comprises a sequence of any one of SEQ IDNOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291,2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302 or asequence set forth in Table 27; and wherein the antibody is a monoclonalantibody, a polyclonal antibody, a bi-specific antibody, a multispecificantibody, a grafted antibody, a human antibody, a humanized antibody, asynthetic antibody, a chimeric antibody, a camelized antibody, asingle-chain Fvs (scFv), a single chain antibody, a Fab fragment, aF(ab′)₂ fragment, a Fd fragment, a Fv fragment, a single-domainantibody, an isolated complementarity determining region (CDR), adiabody, a fragment comprised of only a single monomeric variabledomain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic(anti-Id) antibody, or ab antigen-binding fragments thereof.

Provided herein are antagonists of GLP1R comprising SEQ ID NO: 2279 or2320. Further provided herein are antagonists of GLP1R, wherein theantagonist comprises an EC50 of no more than 1.5 nM. Further providedherein are antagonists of GLP1R, wherein the antagonist comprises anEC50 of no more than 1.0 nM. Further provided herein are antagonists ofGLP1R, wherein the antagonist comprises an EC50 of no more than 0.5 nM.Further provided herein are antagonists of GLP1R, wherein the antagonistis an antibody or antibody fragment.

Provided herein are agonists of GLP1R comprising SEQ ID NO: 2317.Further provided herein are agonists of GLP1R, wherein the agonistcomprises an EC50 of no more than 1.5 nM. Further provided herein areagonists of GLP1R, wherein the agonist comprises an EC50 of no more than1.0 nM. Further provided herein are agonists of GLP1R, wherein theagonist comprises an EC50 of no more than 0.5 nM. Further providedherein are agonists of GLP1R, wherein the agonist is an antibody orantibody fragment.

Provided herein are methods of inhibiting GLP1R activity, comprisingadministering the antibody or antibody fragment as described herein.Further provided herein are methods of inhibiting GLP1R activity,wherein the antibody or antibody fragment is an allosteric modulator.Further provided herein are methods of inhibiting GLP1R activity,wherein the antibody or antibody fragment is a negative allostericmodulator.

Provided herein are methods for treatment of a metabolic disorder,comprising administering to a subject in need thereof the antibody asdescribed herein. Provided herein are methods for treatment of ametabolic disorder, wherein the metabolic disorder is Type II diabetesor obesity.

Provided herein are protein libraries encoded by the nucleic acidlibrary as described herein, wherein the protein library comprisespeptides. Further provided herein are protein libraries, wherein theprotein library comprises immunoglobulins. Further provided herein areprotein libraries, wherein the protein library comprises antibodies.Further provided herein are protein libraries, wherein the proteinlibrary is a peptidomimetic library.

Provided herein are vector libraries comprising the nucleic acid libraryas described herein. Provided herein are cell libraries comprising thenucleic acid library as described herein. Provided herein are celllibraries comprising the protein library as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a first schematic of an immunoglobulin scaffold.

FIG. 1B depicts a second schematic of an immunoglobulin scaffold.

FIG. 2 depicts a schematic of a motif for placement in a scaffold.

FIG. 3 presents a diagram of steps demonstrating an exemplary processworkflow for gene synthesis as disclosed herein.

FIG. 4 illustrates an example of a computer system.

FIG. 5 is a block diagram illustrating an architecture of a computersystem.

FIG. 6 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 7 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

FIG. 8A depicts a schematic of an immunoglobulin scaffold comprising aVH domain attached to a VL domain using a linker.

FIG. 8B depicts a schematic of a full-domain architecture of animmunoglobulin scaffold comprising a VH domain attached to a VL domainusing a linker, a leader sequence, and pIII sequence.

FIG. 8C depicts a schematic of four framework elements (FW1, FW2, FW3,FW4) and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.

FIGS. 9A-90 depict the cell binding data for GLP1R-2 (FIG. 9A), GLP1R-3(FIG. 9B), GLP1R-8 (FIG. 9C), GLP1R-26 (FIG. 9D), GLP1R-30 (FIG. 9E),GLP1R-56 (FIG. 9F), GLP1R-58 (FIG. 9G), GLP1R-10 (FIG. 9H), GLP1R-25(FIG. 9I), GLP1R-60 (FIG. 9J), GLP1R-70 (FIG. 9K), GLP1R-72 (FIG. 9L),GLP1R-83 (FIG. 9M), GLP1R-93 (FIG. 9N), and GLP1R-98 (FIG. 9O).

FIGS. 10A-100 depict graphs of GLP1R-2 (FIG. 10A), GLP1R-3 (FIG. 10B),GLP1R-8 (FIG. 10C), GLP1R-26 (FIG. 10D), GLP1R-30 (FIG. 10E), GLP1R-56(FIG. 10F), GLP1R-58 (FIG. 10G), GLP1R-10 (FIG. 10H), GLP1R-25 (FIG.10I), GLP1R-60 (FIG. 10J), GLP1R-70 (FIG. 10K), GLP1R-72 (FIG. 10L),GLP1R-83 (FIG. 10M), GLP1R-93 (FIG. 10N), and GLP1R-98 (FIG. 10O)variants on inhibition of GLP1-7-36 peptide induced cAMP activity.

FIGS. 11A-11G depict cell functional data for GLP1R-2 (FIG. 11A),GLP1R-3 (FIG. 11B), GLP1R-8 (FIG. 11C), GLP1R-26 (FIG. 11D), GLP1R-30(FIG. 11E), GLP1R-56 (FIG. 11F), and GLP1R-58 (FIG. 11G).

FIGS. 12A-12G depict graphs of GLP1R-2 (FIG. 12A), GLP1R-3 (FIG. 12B),GLP1R-8 (FIG. 12C), GLP1R-26 (FIG. 12D), GLP1R-30 (FIG. 12E), GLP1R-56(FIG. 12F), and GLP1R-58 (FIG. 12G) variants on inhibition of Exendin-4peptide induced cAMP activity.

FIG. 13 depicts a schematic of glucagon (SEQ ID NO: 2740), GLP1-1 (SEQID NO: 6), and (GLP-2 SEQ ID NO: 2741).

FIGS. 14A-14C depict cell-binding affinity of purified immunoglobulins.

FIG. 14D depicts cAMP activity of purified immunoglobulins.

FIGS. 15A-15H depict binding curves plotting IgG concentrations innanomolar (nM) against MFI (mean fluorescence intensity) for GLP1R-238(FIG. 15A), GLP1R-240 (FIG. 15B), GLP1R-241 (FIG. 15C), GLP1R-242 (FIG.15D), GLP1R-243 (FIG. 15E), GLP1R-244 (FIG. 15F), pGPCR-GLP1R-43 (FIG.15G), and pGPCR-GLP1R-44 (FIG. 15H).

FIGS. 16A-161 depict flow cytometry data of binding assays presented asdot plots with 100 nM IgG of GLP1R-238 (FIG. 16A), GLP1R-240 (FIG. 16B),GLP1R-241 (FIG. 16C), GLP1R-242 (FIG. 16D), GLP1R-243 (FIG. 16E),GLP1R-244 (FIG. 16F), pGPCR-GLP1R-43 (FIG. 16G), pGPCR-GLP1R-44 (FIG.16H), and GLP1R-239 (FIG. 16I).

FIGS. 17A-17B depict data from cAMP assays with relative luminescenceunits (RLU) on the y-axis and concentration in nanomolar (nM) on thex-axis. cAMP was measured in response to GLP1 (7-36), GLP1R-238,GLP1R-239, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, GLP1R-244,pGPCR-GLP1R-43, pGPCR-GLP1R-44, and buffer.

FIG. 17C depicts a graph of cAMP allosteric effect of GLP1R-241.

FIG. 17D depicts a graph of beta-arrestin recruitment of GLP1R-241.

FIG. 17E depicts a graph of GLP1R-241 internalization.

FIGS. 18A-18B depict data from cAMP assays with relative luminescenceunits (RLU) on the y-axis and concentration of GLP1 (7-36) in nanomolar(nM) on the x-axis. Allosteric effects of GLP1R-238, GLP1R-239,GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, GLP1R-244, pGPCR-GLP1R-43,pGPCR-GLP1R-44, and no antibody were tested.

FIGS. 19A-19F depict flow cytometry data of binding assays presented asdot plots and histograms for GLP1R-59-2 (FIG. 19A), GLP1R-59-241 (FIG.19B), GLP1R-59-243 (FIG. 19C), GLP1R-3 (FIG. 19D), GLP1R-241 (FIG. 19E),and GLP1R-2 (FIG. 19F). FIGS. 19A-19F also depict titration curvesplotting IgG concentrations in nanomolar (nM) against MFI (meanfluorescence intensity) for GLP1R-59-2 (FIG. 19A), GLP1R-59-241 (FIG.19B), GLP1R-59-243 (FIG. 19C), GLP1R-3 (FIG. 19D), GLP1R-241 (FIG. 19E),and GLP1R-2 (FIG. 19F).

FIGS. 20A-20F depict data from cAMP assays with relative luminescenceunits (RLU) on the y-axis and concentration of GLP1 (7-36) in nanomolar(nM) on the x-axis as well as beta-arrestin recruitment and receptorinternalization for GLP1R-59-2 (FIG. 20A), GLP1R-59-241 (FIG. 20B),GLP1R-59-243 (FIG. 20C), GLP1R-3 (FIG. 20D), GLP1R-241 (FIG. 20E), andGLP1R-2 (FIG. 20F).

FIGS. 21A-21B depicts graphs of TIGIT affinity distribution for the VHHlibraries, depicting either the affinity threshold from 20 to 4000 (FIG.21A) or the affinity threshold from 20 to 1000 (FIG. 21B). Out of 140VHH binders, 51 variants were <100 nM and 90 variants were <200 nM.

FIGS. 22A-22B depict graphs of FACs analysis (FIG. 22A) and graphs of adose curve and specificity (FIG. 22B) of GLP1R-43-77.

FIG. 23A depicts a schema of heavy chain IGHV3-23 design. FIG. 23 Adiscloses SEQ ID NOS 2742-2747, respectively, in order of appearance.

FIG. 23B depicts a schema of heavy chain IGHV1-69 design. FIG. 23Bdiscloses SEQ ID NOS 2748-2753, respectively, in order of appearance.

FIG. 23C depicts a schema of light chains IGKV 2-28 and IGLV 1-51design. FIG. 23C discloses SEQ ID NOS 2754-2759, respectively, in orderof appearance.

FIG. 23D depicts a schema of the theoretical diversity and finaldiversity of a GLP1R library.

FIGS. 23E-23F depict graphs of FACS binding of GLP1R IgGs.

FIGS. 23G-23H depict graphs of cAMP assays using purified GLP1R IgGs.

FIG. 24A depicts a graph of GLP1R-3 inhibition as compared to noantibody. Relative luminescence units (RLU) is depicted on the y-axis,and concentration of GLP1 (7-36) is depicted in nanomolar (nM) on thex-axis.

FIG. 24B depicts a graph of GLP1R-3 inhibition at high concentrationsfollowing stimulation with 0.05 nM GLP1 (7-36). Relative luminescenceunits (RLU) is depicted on the y-axis, and concentration of GLP1R-3 isdepicted in nanomolar (nM) on the x-axis.

FIG. 24C depicts glucose levels after glucose administration whentreated with vehicle (triangles), liraglutide (squares), and GLP1R-3(circles) in a mouse model of diet induced obesity.

FIG. 24D depicts glucose levels after glucose administration whentreated with vehicle (open triangles), liraglutide (squares), andGLP1R-59-2 (closed triangles) in a mouse model of diet induced obesity.

FIG. 25A depicts a graph of the blood glucose levels in mice (mg/dL;y-axis) treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist), andcontrol over time (in minutes, x-axis).

FIG. 25B depicts a graph of blood glucose levels in mice (mg/dL; y-axis)treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control.

FIG. 25C depicts a graph of the blood glucose levels (mg/dL; y-axis) inGLP1R-59-2 (agonist) treated mice in both the fasted (p=0.0008) andnon-fasted (p<0.0001) mice compared to control.

FIG. 25D depicts a graph of the blood glucose levels (mg/dL/min; y-axis)in pre-dosed GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and controlmice.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventionalmolecular biology techniques, which are within the skill of the art.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

Definitions

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

Unless specifically stated, as used herein, the term “nucleic acid”encompasses double- or triple-stranded nucleic acids, as well assingle-stranded molecules. In double- or triple-stranded nucleic acids,the nucleic acid strands need not be coextensive (i.e., adouble-stranded nucleic acid need not be double-stranded along theentire length of both strands). Nucleic acid sequences, when provided,are listed in the 5′ to 3′ direction, unless stated otherwise. Methodsdescribed herein provide for the generation of isolated nucleic acids.Methods described herein additionally provide for the generation ofisolated and purified nucleic acids. A “nucleic acid” as referred toherein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, or more bases in length. Moreover, providedherein are methods for the synthesis of any number ofpolypeptide-segments encoding nucleotide sequences, including sequencesencoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomalpeptide-synthetase (NRPS) modules and synthetic variants, polypeptidesegments of other modular proteins, such as antibodies, polypeptidesegments from other protein families, including non-coding DNA or RNA,such as regulatory sequences e.g. promoters, transcription factors,enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived frommicroRNA, or any functional or structural DNA or RNA unit of interest.The following are non-limiting examples of polynucleotides: coding ornon-coding regions of a gene or gene fragment, intergenic DNA, loci(locus) defined from linkage analysis, exons, introns, messenger RNA(mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA,ribozymes, complementary DNA (cDNA), which is a DNA representation ofmRNA, usually obtained by reverse transcription of messenger RNA (mRNA)or by amplification; DNA molecules produced synthetically or byamplification, genomic DNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. cDNAencoding for a gene or gene fragment referred herein may comprise atleast one region encoding for exon sequences without an interveningintron sequence in the genomic equivalent sequence.

GPCR Libraries for GLP1 Receptor

Provided herein are methods and compositions relating to Gprotein-coupled receptor (GPCR) binding libraries for glucagon-likepeptide-1 receptor (GLP1R) comprising nucleic acids encoding for ascaffold comprising a GPCR binding domain. Scaffolds as described hereincan stably support a GPCR binding domain. The GPCR binding domain may bedesigned based on surface interactions of a GLP1R ligand and GLP1R.Libraries as described herein may be further variegated to provide forvariant libraries comprising nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence. Further described herein are protein libraries that maybe generated when the nucleic acid libraries are translated. In someinstances, nucleic acid libraries as described herein are transferredinto cells to generate a cell library. Also provided herein aredownstream applications for the libraries synthesized using methodsdescribed herein. Downstream applications include identification ofvariant nucleic acids or protein sequences with enhanced biologicallyrelevant functions, e.g., improved stability, affinity, binding,functional activity, and for the treatment or prevention of a diseasestate associated with GPCR signaling.

Scaffold Libraries

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein sequences for GPCR binding domains are placed in thescaffold. Scaffold described herein allow for improved stability for arange of GPCR binding domain encoding sequences when inserted into thescaffold, as compared to an unmodified scaffold. Exemplary scaffoldsinclude, but are not limited to, a protein, a peptide, animmunoglobulin, derivatives thereof, or combinations thereof. In someinstances, the scaffold is an immunoglobulin. Scaffolds as describedherein comprise improved functional activity, structural stability,expression, specificity, or a combination thereof. In some instances,scaffolds comprise long regions for supporting a GPCR binding domain.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein the scaffold is an immunoglobulin. In some instances,the immunoglobulin is an antibody. As used herein, the term antibodywill be understood to include proteins having the characteristictwo-armed, Y-shape of a typical antibody molecule as well as one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Exemplary antibodies include, but are not limited to, amonoclonal antibody, a polyclonal antibody, a bi-specific antibody, amultispecific antibody, a grafted antibody, a human antibody, ahumanized antibody, a synthetic antibody, a chimeric antibody, acamelized antibody, a single-chain Fvs (scFv) (including fragments inwhich the VL and VH are joined using recombinant methods by a syntheticor natural linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules,including single chain Fab and scFab), a single chain antibody, a Fabfragment (including monovalent fragments comprising the VL, VH, CL, andCH1 domains), a F(ab′)₂ fragment (including bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion), a Fd fragment (including fragments comprising the VH and CH1fragment), a Fv fragment (including fragments comprising the VL and VHdomains of a single arm of an antibody), a single-domain antibody (dAbor sdAb) (including fragments comprising a VH domain), an isolatedcomplementarity determining region (CDR), a diabody (including fragmentscomprising bivalent dimers such as two VL and VH domains bound to eachother and recognizing two different antigens), a fragment comprised ofonly a single monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof. In some instances, the libraries disclosed hereincomprise nucleic acids encoding for a scaffold, wherein the scaffold isa Fv antibody, including Fv antibodies comprised of the minimum antibodyfragment which contains a complete antigen-recognition andantigen-binding site. In some embodiments, the Fv antibody consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association, and the three hypervariable regions of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. In some embodiments, the six hypervariableregions confer antigen-binding specificity to the antibody. In someembodiments, a single variable domain (or half of an Fv comprising onlythree hypervariable regions specific for an antigen, including singledomain antibodies isolated from camelid animals comprising one heavychain variable domain such as VHH antibodies or nanobodies) has theability to recognize and bind antigen. In some instances, the librariesdisclosed herein comprise nucleic acids encoding for a scaffold, whereinthe scaffold is a single-chain Fv or scFv, including antibody fragmentscomprising a VH, a VL, or both a VH and VL domain, wherein both domainsare present in a single polypeptide chain. In some embodiments, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains allowing the scFv to form the desired structure for antigenbinding. In some instances, a scFv is linked to the Fc fragment or a VHHis linked to the Fc fragment (including minibodies). In some instances,the antibody comprises immunoglobulin molecules and immunologicallyactive fragments of immunoglobulin molecules, e.g., molecules thatcontain an antigen binding site. Immunoglobulin molecules are of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2,IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.

In some embodiments, libraries comprise immunoglobulins that are adaptedto the species of an intended therapeutic target. Generally, thesemethods include “mammalization” and comprises methods for transferringdonor antigen-binding information to a less immunogenic mammal antibodyacceptor to generate useful therapeutic treatments. In some instances,the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee,baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, andhuman. In some instances, provided herein are libraries and methods forfelinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodiesthat contain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human antibody (recipient antibody) inwhich residues from one or more CDRs are replaced by residues from oneor more CDRs of a non-human antibody (donor antibody). The donorantibody can be any suitable non-human antibody, such as a mouse, rat,rabbit, chicken, or non-human primate antibody having a desiredspecificity, affinity, or biological effect. In some instances, selectedframework region residues of the recipient antibody are replaced by thecorresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found ineither the recipient antibody or the donor antibody. In some instances,these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canineantigen-binding information from a donor antibody to a less immunogeniccanine antibody acceptor to generate treatments useful as therapeuticsin dogs. In some instances, caninized forms of non-canine antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-canine antibodies. In some instances, caninizedantibodies are canine antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-canine species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the canine antibody are replaced by corresponding non-canineFR residues. In some instances, caninized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The caninized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a canineantibody.

“Felinization” can comprise a method for transferring non-felineantigen-binding information from a donor antibody to a less immunogenicfeline antibody acceptor to generate treatments useful as therapeuticsin cats. In some instances, felinized forms of non-feline antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-feline antibodies. In some instances, felinizedantibodies are feline antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-feline species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the feline antibody are replaced by corresponding non-felineFR residues. In some instances, felinized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The felinized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a felinizeantibody.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein the scaffold is a non-immunoglobulin. In someinstances, the scaffold is a non-immunoglobulin binding domain. Forexample, the scaffold is an antibody mimetic. Exemplary antibodymimetics include, but are not limited to, anticalins, affilins, affibodymolecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins,fynomers, Kunitz domain-based proteins, monobodies, anticalins,knottins, armadillo repeat protein-based proteins, and bicyclicpeptides.

Libraries described herein comprising nucleic acids encoding for ascaffold, wherein the scaffold is an immunoglobulin, comprise variationsin at least one region of the immunoglobulin. Exemplary regions of theantibody for variation include, but are not limited to, acomplementarity-determining region (CDR), a variable domain, or aconstant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. Insome instances, the CDR is a heavy domain including, but not limited to,CDR-H1, CDR-H2, and CDR-H3. In some instances, the CDR is a light domainincluding, but not limited to, CDR-L1, CDR-L2, and CDR-L3. In someinstances, the variable domain is variable domain, light chain (VL) orvariable domain, heavy chain (VH). In some instances, the VL domaincomprises kappa or lambda chains. In some instances, the constant domainis constant domain, light chain (CL) or constant domain, heavy chain(CH).

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for a scaffold, wherein each nucleic acid encodesfor a predetermined variant of at least one predetermined referencenucleic acid sequence. In some cases, the predetermined referencesequence is a nucleic acid sequence encoding for a protein, and thevariant library comprises sequences encoding for variation of at least asingle codon such that a plurality of different variants of a singleresidue in the subsequent protein encoded by the synthesized nucleicacid are generated by standard translation processes. In some instances,the scaffold library comprises varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon of a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, orVH domain. In some instances, the variant library comprises sequencesencoding for variation of multiple codons of a CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the variantlibrary comprises sequences encoding for variation of multiple codons offramework element 1 (FW1), framework element 2 (FW2), framework element3 (FW3), or framework element 4 (FW4). An exemplary number of codons forvariation include, but are not limited to, at least or about 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the immunoglobulin forvariation is from heavy chain V-gene family, heavy chain D-gene family,heavy chain J-gene family, light chain V-gene family, or light chainJ-gene family. See FIGS. 1A-1B. In some instances, the light chainV-gene family comprises immunoglobulin kappa (IGK) gene orimmunoglobulin lambda (IGL). Exemplary genes include, but are notlimited to, IGHV1-18, IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23,IGHV3-30/33rn, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61,IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1,IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1. In some instances, the geneis IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, orIGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30. In someinstances, the gene is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1.In some instances, the gene is IGHJ3, IGHJ6, IGHJ, or IGHJ4.

Provided herein are libraries comprising nucleic acids encoding forimmunoglobulin scaffolds, wherein the libraries are synthesized withvarious numbers of fragments. In some instances, the fragments comprisethe CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. Insome instances, the fragments comprise framework element 1 (FW1),framework element 2 (FW2), framework element 3 (FW3), or frameworkelement 4 (FW4). In some instances, the scaffold libraries aresynthesized with at least or about 2 fragments, 3 fragments, 4fragments, 5 fragments, or more than 5 fragments. The length of each ofthe nucleic acid fragments or average length of the nucleic acidssynthesized may be at least or about 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, or more than 600 base pairs. In some instances, the length isabout 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 basepairs.

Libraries comprising nucleic acids encoding for immunoglobulin scaffoldsas described herein comprise various lengths of amino acids whentranslated. In some instances, the length of each of the amino acidfragments or average length of the amino acid synthesized may be atleast or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, ormore than 150 amino acids. In some instances, the length of the aminoacid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to100, or 75 to 95 amino acids. In some instances, the length of the aminoacid is about 22 amino acids to about 75 amino acids. In some instances,the immunoglobulin scaffolds comprise at least or about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than5000 amino acids.

A number of variant sequences for the at least one region of theimmunoglobulin for variation are de novo synthesized using methods asdescribed herein. In some instances, a number of variant sequences is denovo synthesized for CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL,VH, or combinations thereof. In some instances, a number of variantsequences is de novo synthesized for framework element 1 (FW1),framework element 2 (FW2), framework element 3 (FW3), or frameworkelement 4 (FW4). The number of variant sequences may be at least orabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, or more than 500 sequences. In some instances, thenumber of variant sequences is at least or about 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000sequences. In some instances, the number of variant sequences is about10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325sequences.

Variant sequences for the at least one region of the immunoglobulin, insome instances, vary in length or sequence. In some instances, the atleast one region that is de novo synthesized is for CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or combinations thereof. In someinstances, the at least one region that is de novo synthesized is forframework element 1 (FW1), framework element 2 (FW2), framework element3 (FW3), or framework element 4 (FW4). In some instances, the variantsequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, or more than 50 variant nucleotides or aminoacids as compared to wild-type. In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, or 50 additional nucleotides or amino acids as comparedto wild-type. In some instances, the variant sequence comprises at leastor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50 less nucleotides or amino acids as compared to wild-type. In someinstances, the libraries comprise at least or about 10¹, 10², 10³ 10⁴10⁵ 10⁶, 10⁷ 10⁸, 10⁹ 10¹⁰ or more than 10¹⁰ variants.

Following synthesis of scaffold libraries, scaffold libraries may beused for screening and analysis. For example, scaffold libraries areassayed for library displayability and panning. In some instances,displayability is assayed using a selectable tag. Exemplary tagsinclude, but are not limited to, a radioactive label, a fluorescentlabel, an enzyme, a chemiluminescent tag, a colorimetric tag, anaffinity tag or other labels or tags that are known in the art. In someinstances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA),or FLAG. In some instances, scaffold libraries are assayed by sequencingusing various methods including, but not limited to, single-moleculereal-time (SMRT) sequencing, Polony sequencing, sequencing by ligation,reversible terminator sequencing, proton detection sequencing, ionsemiconductor sequencing, nanopore sequencing, electronic sequencing,pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g.,Sanger) sequencing, +S sequencing, or sequencing by synthesis.

In some instances, the scaffold libraries are assayed for functionalactivity, structural stability (e.g., thermal stable or pH stable),expression, specificity, or a combination thereof. In some instances,the scaffold libraries are assayed for scaffolds capable of folding. Insome instances, a region of the antibody is assayed for functionalactivity, structural stability, expression, specificity, folding, or acombination thereof. For example, a VH region or VL region is assayedfor functional activity, structural stability, expression, specificity,folding, or a combination thereof.

GLP1R Libraries

Provided herein are GLP1R binding libraries comprising nucleic acidsencoding for scaffolds comprising sequences for GLP1R binding domains.In some instances, the scaffolds are immunoglobulins. In some instances,the scaffolds comprising sequences for GLP1R binding domains aredetermined by interactions between the GLP1R binding domains and theGLP1R.

Provided herein are libraries comprising nucleic acids encodingscaffolds comprising GLP1R binding domains, wherein the GLP1R bindingdomains are designed based on surface interactions on GLP1R. In someinstances, the GLP1R comprises a sequence as defined by SEQ ID NO: 1. Insome instances, the GLP1R binding domains interact with the amino- orcarboxy-terminus of the GLP1R. In some instances, the GLP1R bindingdomains interact with at least one transmembrane domain including, butnot limited to, transmembrane domain 1 (TM1), transmembrane domain 2(TM2), transmembrane domain 3 (TM3), transmembrane domain 4 (TM4),transmembrane domain 5 (TM5), transmembrane domain 6 (TM6), andtransmembrane domain 7 (TM7). In some instances, the GLP1R bindingdomains interact with an intracellular surface of the GLP1R. Forexample, the GLP1R binding domains interact with at least oneintracellular loop including, but not limited to, intracellular loop 1(ICL1), intracellular loop 2 (ICL2), and intracellular loop 3 (ICL3). Insome instances, the GLP1R binding domains interact with an extracellularsurface of the GLP1R. For example, the GLP1R binding domains interactwith at least one extracellular domain (ECD) or extracellular loop (ECL)of the GLP1R. The extracellular loops include, but are not limited to,extracellular loop 1 (ECL1), extracellular loop 2 (ECL2), andextracellular loop 3 (ECL3).

Described herein are GLP1R binding domains, wherein the GLP1R bindingdomains are designed based on surface interactions between a GLP1Rligand and the GLP1R. In some instances, the ligand is a peptide. Insome instances, the ligand is glucagon, glucagon-like peptide 1-(7-36)amide, glucagon-like peptide 1-(7-37), liraglutide, exendin-4,lixisenatide, T-0632, GLP1R0017, or BETP. In some instances, the ligandis a GLP1R agonist. In some instances, the ligand is a GLP1R antagonist.In some instances, the ligand is a GLP1R allosteric modulator. In someinstances, the allosteric modulator is a negative allosteric modulator.In some instances, the allosteric modulator is a positive allostericmodulator.

Sequences of GLP1R binding domains based on surface interactions betweena GLP1R ligand and the GLP1R are analyzed using various methods. Forexample, multispecies computational analysis is performed. In someinstances, a structure analysis is performed. In some instances, asequence analysis is performed. Sequence analysis can be performed usinga database known in the art. Non-limiting examples of databases include,but are not limited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi),UCSC Genome Browser (genome.ucsc.edu/), UniProt (www.uniprot.org/), andIUPHAR/BPS Guide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are GLP1R binding domains designed based on sequenceanalysis among various organisms. For example, sequence analysis isperformed to identify homologous sequences in different organisms.Exemplary organisms include, but are not limited to, mouse, rat, equine,sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan,monkey), dog, cat, pig, donkey, rabbit, fish, fly, and human.

Following identification of GLP1R binding domains, libraries comprisingnucleic acids encoding for the GLP1R binding domains may be generated.In some instances, libraries of GLP1R binding domains comprise sequencesof GLP1R binding domains designed based on conformational ligandinteractions, peptide ligand interactions, small molecule ligandinteractions, extracellular domains of GLP1R, or antibodies that targetGLP1R. In some instances, libraries of GLP1R binding domains comprisesequences of GLP1R binding domains designed based on peptide ligandinteractions. Libraries of GLP1R binding domains may be translated togenerate protein libraries. In some instances, libraries of GLP1Rbinding domains are translated to generate peptide libraries,immunoglobulin libraries, derivatives thereof, or combinations thereof.In some instances, libraries of GLP1R binding domains are translated togenerate protein libraries that are further modified to generatepeptidomimetic libraries. In some instances, libraries of GLP1R bindingdomains are translated to generate protein libraries that are used togenerate small molecules.

Methods described herein provide for synthesis of libraries of GLP1Rbinding domains comprising nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence. In some cases, the predetermined reference sequence is anucleic acid sequence encoding for a protein, and the variant librarycomprises sequences encoding for variation of at least a single codonsuch that a plurality of different variants of a single residue in thesubsequent protein encoded by the synthesized nucleic acid are generatedby standard translation processes. In some instances, the libraries ofGLP1R binding domains comprise varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon in a GLP1R binding domain. In some instances, the variantlibrary comprises sequences encoding for variation of multiple codons ina GLP1R binding domain. An exemplary number of codons for variationinclude, but are not limited to, at least or about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for the GLP1R binding domains, wherein thelibraries comprise sequences encoding for variation of length of theGLP1R binding domains. In some instances, the library comprisessequences encoding for variation of length of at least or about 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less ascompared to a predetermined reference sequence. In some instances, thelibrary comprises sequences encoding for variation of length of at leastor about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or morethan 300 codons more as compared to a predetermined reference sequence.

Following identification of GLP1R binding domains, the GLP1R bindingdomains may be placed in scaffolds as described herein. In someinstances, the scaffolds are immunoglobulins. In some instances, theGLP1R binding domains are placed in the CDR-H3 region. GPCR bindingdomains that may be placed in scaffolds can also be referred to as amotif. Scaffolds comprising GLP1R binding domains may be designed basedon binding, specificity, stability, expression, folding, or downstreamactivity. In some instances, the scaffolds comprising GLP1R bindingdomains enable contact with the GLP1R. In some instances, the scaffoldscomprising GLP1R binding domains enables high affinity binding with theGLP1R. An exemplary amino acid sequence of GLP1R binding domain isdescribed in Table 1.

TABLE 1 GLP1R amino acid sequences SEQ ID NO GPCR Amino Acid Sequence 1GLP1R RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFLYIIYTVGYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSVLSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTS SLSSGATAGSSMYTATCQASCS

Provided herein are scaffolds comprising GLP1R binding domains, whereinthe sequences of the GLP1R binding domains support interaction withGLP1R. The sequence may be homologous or identical to a sequence of aGLP1R ligand. In some instances, the GLP1R binding domain sequencecomprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In someinstances, the GLP1R binding domain sequence comprises at least or about95% homology to SEQ ID NO: 1. In some instances, the GLP1R bindingdomain sequence comprises at least or about 97% homology to SEQ IDNO: 1. In some instances, the GLP1R binding domain sequence comprises atleast or about 99% homology to SEQ ID NO: 1. In some instances, theGLP1R binding domain sequence comprises at least or about 100% homologyto SEQ ID NO: 1. In some instances, the GLP1R binding domain sequencecomprises at least a portion having at least or about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQID NO: 1.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity.

The term “homology” or “similarity” between two proteins is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one protein sequence to the second protein sequence.Similarity may be determined by procedures which are well-known in theart, for example, a BLAST program (Basic Local Alignment Search Tool atthe National Center for Biological Information).

Provided herein are GLP1R binding libraries comprising nucleic acidsencoding for scaffolds comprising GLP1R binding domains comprisevariation in domain type, domain length, or residue variation. In someinstances, the domain is a region in the scaffold comprising the GLP1Rbinding domains. For example, the region is the VH, CDR-H3, or VLdomain. In some instances, the domain is the GLP1R binding domain.

Methods described herein provide for synthesis of a GLP1R bindinglibrary of nucleic acids each encoding for a predetermined variant of atleast one predetermined reference nucleic acid sequence. In some cases,the predetermined reference sequence is a nucleic acid sequence encodingfor a protein, and the variant library comprises sequences encoding forvariation of at least a single codon such that a plurality of differentvariants of a single residue in the subsequent protein encoded by thesynthesized nucleic acid are generated by standard translationprocesses. In some instances, the GLP1R binding library comprises variednucleic acids collectively encoding variations at multiple positions. Insome instances, the variant library comprises sequences encoding forvariation of at least a single codon of a VH, CDR-H3, or VL domain. Insome instances, the variant library comprises sequences encoding forvariation of at least a single codon in a GLP1R binding domain. Forexample, at least one single codon of a GLP1R binding domain as listedin Table 1 is varied. In some instances, the variant library comprisessequences encoding for variation of multiple codons of a VH, CDR-H3, orVL domain. In some instances, the variant library comprises sequencesencoding for variation of multiple codons in a GLP1R binding domain. Anexemplary number of codons for variation include, but are not limitedto, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, ormore than 300 codons.

Methods described herein provide for synthesis of a GLP1R bindinglibrary of nucleic acids each encoding for a predetermined variant of atleast one predetermined reference nucleic acid sequence, wherein theGLP1R binding library comprises sequences encoding for variation oflength of a domain. In some instances, the domain is VH, CDR-H3, or VLdomain. In some instances, the domain is the GLP1R binding domain. Insome instances, the library comprises sequences encoding for variationof length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275,300, or more than 300 codons less as compared to a predeterminedreference sequence. In some instances, the library comprises sequencesencoding for variation of length of at least or about 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200, 225, 250, 275, 300, or more than 300 codons more ascompared to a predetermined reference sequence.

Provided herein are GLP1R binding libraries comprising nucleic acidsencoding for scaffolds comprising GLP1R binding domains, wherein theGLP1R binding libraries are synthesized with various numbers offragments. In some instances, the fragments comprise the VH, CDR-H3, orVL domain. In some instances, the GLP1R binding libraries aresynthesized with at least or about 2 fragments, 3 fragments, 4fragments, 5 fragments, or more than 5 fragments. The length of each ofthe nucleic acid fragments or average length of the nucleic acidssynthesized may be at least or about 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, or more than 600 base pairs. In some instances, the length isabout 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 basepairs.

GLP1R binding libraries comprising nucleic acids encoding for scaffoldscomprising GLP1R binding domains as described herein comprise variouslengths of amino acids when translated. In some instances, the length ofeach of the amino acid fragments or average length of the amino acidsynthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, or more than 150 amino acids. In some instances, thelength of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110,65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, thelength of the amino acid is about 22 to about 75 amino acids.

GLP1R binding libraries comprising de novo synthesized variant sequencesencoding for scaffolds comprising GLP1R binding domains comprise anumber of variant sequences. In some instances, a number of variantsequences is de novo synthesized for a CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, CDR-L3, VL, VH, or a combination thereof. In some instances, anumber of variant sequences is de novo synthesized for framework element1 (FW1), framework element 2 (FW2), framework element 3 (FW3), orframework element 4 (FW4). In some instances, a number of variantsequences is de novo synthesized for a GPCR binding domain. For example,the number of variant sequences is about 1 to about 10 sequences for theVH domain, about 10⁸ sequences for the GLP1R binding domain, and about 1to about 44 sequences for the VK domain. The number of variant sequencesmay be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. Insome instances, the number of variant sequences is about 10 to 300, 25to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150 sequences.

GLP1R binding libraries comprising de novo synthesized variant sequencesencoding for scaffolds comprising GLP1R binding domains compriseimproved diversity. For example, variants are generated by placing GLP1Rbinding domain variants in immunoglobulin scaffold variants comprisingN-terminal CDR-H3 variations and C-terminal CDR-H3 variations. In someinstances, variants include affinity maturation variants. Alternativelyor in combination, variants include variants in other regions of theimmunoglobulin including, but not limited to, CDR-H1, CDR-H2, CDR-L1,CDR-L2, and CDR-L3. In some instances, the number of variants of theGLP1R binding libraries is least or about 10⁴ 10⁵ 10⁶, 10⁷ 10⁸, 10⁹ 10¹⁰10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than10²⁰ non-identical sequences. For example, a library comprising about 10variant sequences for a VH region, about 237 variant sequences for aCDR-H3 region, and about 43 variant sequences for a VL and CDR-L3 regioncomprises 10⁵ non-identical sequences (10×237×43).

Provided herein are libraries comprising nucleic acids encoding for aGLP1R antibody comprising variation in at least one region of theantibody, wherein the region is the CDR region. In some instances, theGLP1R antibody is a single domain antibody comprising one heavy chainvariable domain such as a VHH antibody. In some instances, the VHHantibody comprises variation in one or more CDR regions. In someinstances, libraries described herein comprise at least or about 1, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or morethan 3000 sequences of a CDR1, CDR2, or CDR3. In some instances,libraries described herein comprise at least or about 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸,10¹⁹, 10²⁰ or more than 10²⁰ sequences of a CDR1, CDR2, or CDR3. Forexample, the libraries comprise at least 2000 sequences of a CDR1, atleast 1200 sequences for CDR2, and at least 1600 sequences for CDR3. Insome instances, each sequence is non-identical.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain(VL) can be referred to as CDR-L1, CDR-L2, or CDR-L3, respectively. Insome instances, libraries described herein comprise at least or about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000,2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, orCDR3 of the VL. In some instances, libraries described herein compriseat least or about 10⁴ 10⁵ 10⁶, 10⁷ 10⁸, 10⁹ 10¹⁰ 10¹¹ 10¹², 10¹³, 10¹⁴,10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences of aCDR1, CDR2, or CDR3 of the VL. For example, the libraries comprise atleast 20 sequences of a CDR1 of the VL, at least 4 sequences of a CDR2of the VL, and at least 140 sequences of a CDR3 of the VL. In someinstances, the libraries comprise at least 2 sequences of a CDR1 of theVL, at least 1 sequence of CDR2 of the VL, and at least 3000 sequencesof a CDR3 of the VL. In some instances, the VL is IGKV1-39, IGKV1-9,IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14,IGLV1-40, or IGLV3-1. In some instances, the VL is IGKV2-28. In someinstances, the VL is IGLV1-51.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,heavy chain (VH). CDR1, CDR2, or CDR3 of a variable domain, heavy chain(VH) can be referred to as CDR-H1, CDR-H2, or CDR-H3, respectively. Insome instances, libraries described herein comprise at least or about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000,2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, orCDR3 of the VH. In some instances, libraries described herein compriseat least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VH. For example, the libraries comprise atleast 30 sequences of a CDR1 of the VH, at least 570 sequences of a CDR2of the VH, and at least 10⁸ sequences of a CDR3 of the VH. In someinstances, the libraries comprise at least 30 sequences of a CDR1 of theVH, at least 860 sequences of a CDR2 of the VH, and at least 10⁷sequences of a CDR3 of the VH. In some instances, the VH is IGHV1-18,IGHV1 69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74,IGHV4-39, or IGHV4-59/61. In some instances, the VH is IGHV1-69,IGHV3-30, IGHV3-23, IGHV3, IGHV1 46, IGHV3-7, IGHV1, or IGHV1-8. In someinstances, the VH is IGHV1-69 and IGHV3-30. In some instances, the VH isIGHV3-23.

Libraries as described herein, in some embodiments, comprise varyinglengths of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3. In someinstances, the length of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, orCDR-H3 comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,60, 70, 80, 90, or more than 90 amino acids in length. For example, theCDR-H3 comprises at least or about 12, 15, 16, 17, 20, 21, or 23 aminoacids in length. In some instances, the CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, or CDR-H3 comprises a range of about 1 to about 10, about 5 toabout 15, about 10 to about 20, or about 15 to about 30 amino acids inlength.

Libraries comprising nucleic acids encoding for antibodies havingvariant CDR sequences as described herein comprise various lengths ofamino acids when translated. In some instances, the length of each ofthe amino acid fragments or average length of the amino acid synthesizedmay be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, or more than 150 amino acids. In some instances, the length of theamino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105,70 to 100, or 75 to 95 amino acids. In some instances, the length of theamino acid is about 22 amino acids to about 75 amino acids. In someinstances, the antibodies comprise at least or about 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000amino acids.

Ratios of the lengths of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, orCDR-H3 may vary in libraries described herein. In some instances, aCDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 comprising at least orabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90amino acids in length comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or more than 90% of the library. For example, a CDR-H3comprising about 23 amino acids in length is present in the library at40%, a CDR-H3 comprising about 21 amino acids in length is present inthe library at 30%, a CDR-H3 comprising about 17 amino acids in lengthis present in the library at 20%, and a CDR-H3 comprising about 12 aminoacids in length is present in the library at 10%. In some instances, aCDR-H3 comprising about 20 amino acids in length is present in thelibrary at 40%, a CDR-H3 comprising about 16 amino acids in length ispresent in the library at 30%, a CDR-H3 comprising about 15 amino acidsin length is present in the library at 20%, and a CDR-H3 comprisingabout 12 amino acids in length is present in the library at 10%.

Libraries as described herein encoding for a VHH antibody comprisevariant CDR sequences that are shuffled to generate a library with atheoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰sequences. In some instances, the library has a final library diversityof at least or about 10⁷, 10⁸, 10⁹, 10¹⁰ 10¹¹ 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences.

Provided herein are GLP1R binding libraries encoding for animmunoglobulin. In some instances, the GLP1R immunoglobulin is anantibody. In some instances, the GLP1R immunoglobulin is a VHH antibody.In some instances, the GLP1R immunoglobulin comprises a binding affinity(e.g., kD) to GLP1R of less than 1 nM, less than 1.2 nM, less than 2 nM,less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM,less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM.In some instances, the GLP1R immunoglobulin comprises a kD of less than1 nM. In some instances, the GLP1R immunoglobulin comprises a kD of lessthan 1.2 nM. In some instances, the GLP1R immunoglobulin comprises a kDof less than 2 nM. In some instances, the GLP1R immunoglobulin comprisesa kD of less than 5 nM. In some instances, the GLP1R immunoglobulincomprises a kD of less than 10 nM. In some instances, the GLP1Rimmunoglobulin comprises a kD of less than 13.5 nM. In some instances,the GLP1R immunoglobulin comprises a kD of less than 15 nM. In someinstances, the GLP1R immunoglobulin comprises a kD of less than 20 nM.In some instances, the GLP1R immunoglobulin comprises a kD of less than25 nM. In some instances, the GLP1R immunoglobulin comprises a kD ofless than 30 nM.

In some instances, the GLP1R immunoglobulin is a GLP1R agonist. In someinstances, the GLP1R immunoglobulin is a GLP1R antagonist. In someinstances, the GLP1R immunoglobulin is a GLP1R allosteric modulator. Insome instances, the allosteric modulator is a negative allostericmodulator. In some instances, the allosteric modulator is a positiveallosteric modulator. In some instances, the GLP1R immunoglobulinresults in agonistic, antagonistic, or allosteric effects at aconcentration of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM,20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM,140 nM, 160 nM, 180 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM,800 nM, 900 nM, 1000 nM, or more than 1000 nM. In some instances, theGLP1R immunoglobulin is a negative allosteric modulator. In someinstances, the GLP1R immunoglobulin is a negative allosteric modulatorat a concentration of at least or about 0.001, 0.005, 0.01, 0.05, 0.1,0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. In some instances,the GLP1R immunoglobulin is a negative allosteric modulator at aconcentration in a range of about 0.001 to about 100, 0.01 to about 90,about 0.1 to about 80, 1 to about 50, about 10 to about 40 nM, or about1 to about 10 nM. In some instances, the GLP1R immunoglobulin comprisesan EC50 or IC50 of at least or about 0.001, 0.0025, 0.005, 0.01, 0.025,0.05, 0.06, 0.07, 0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6nM. In some instances, the GLP1R immunoglobulin comprises an EC50 orIC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100nM.

Provided herein are GLP1R binding libraries encoding for animmunoglobulin, wherein the immunoglobulin comprises a long half-life.In some instances, the half-life of the GLP1R immunoglobulin is at leastor about 12 hours, 24 hours 36 hours, 48 hours, 60 hours, 72 hours, 84hours, 96 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours,200 hours, or more than 200 hours. In some instances, the half-life ofthe GLP1R immunoglobulin is in a range of about 12 hours to about 300hours, about 20 hours to about 280 hours, about 40 hours to about 240hours, or about 60 hours to about 200 hours.

GLP1R immunoglobulins as described herein may comprise improvedproperties. In some instances, the GLP1R immunoglobulins are monomeric.In some instances, the GLP1R immunoglobulins are not prone toaggregation. In some instances, at least or about 70%, 75%, 80%, 85%,90%, 95%, or 99% of the GLP1R immunoglobulins are monomeric. In someinstances, the GLP1R immunoglobulins are thermostable. In someinstances, the GLP1R immunoglobulins result in reduced non-specificbinding.

Following synthesis of GLP1R binding libraries comprising nucleic acidsencoding scaffolds comprising GLP1R binding domains, libraries may beused for screening and analysis. For example, libraries are assayed forlibrary displayability and panning. In some instances, displayability isassayed using a selectable tag. Exemplary tags include, but are notlimited to, a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a colorimetric tag, an affinity tag or otherlabels or tags that are known in the art. In some instances, the tag ishistidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In someinstances, the GLP1R binding libraries comprises nucleic acids encodingscaffolds comprising GPCR binding domains with multiple tags such asGFP, FLAG, and Lucy as well as a DNA barcode. In some instances,libraries are assayed by sequencing using various methods including, butnot limited to, single-molecule real-time (SMRT) sequencing, Polonysequencing, sequencing by ligation, reversible terminator sequencing,proton detection sequencing, ion semiconductor sequencing, nanoporesequencing, electronic sequencing, pyrosequencing, Maxam-Gilbertsequencing, chain termination (e.g., Sanger) sequencing, +S sequencing,or sequencing by synthesis.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising GLP1R binding domains, wherein the libraries haveimproved specificity, stability, expression, folding, or downstreamactivity. In some instances, libraries described herein are used forscreening and analysis.

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising GLP1R binding domains, wherein the nucleic acidlibraries are used for screening and analysis. In some instances,screening and analysis comprises in vitro, in vivo, or ex vivo assays.Cells for screening include primary cells taken from living subjects orcell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) oreukaryotes (e.g., animals and plants). Exemplary animal cells include,without limitation, those from a mouse, rabbit, primate, and insect. Insome instances, cells for screening include a cell line including, butnot limited to, Chinese Hamster Ovary (CHO) cell line, human embryonickidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In someinstances, nucleic acid libraries described herein may also be deliveredto a multicellular organism. Exemplary multicellular organisms include,without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries or protein libraries encoded thereof describedherein may be screened for various pharmacological or pharmacokineticproperties. In some instances, the libraries are screened using in vitroassays, in vivo assays, or ex vivo assays. For example, in vitropharmacological or pharmacokinetic properties that are screened include,but are not limited to, binding affinity, binding specificity, andbinding avidity. Exemplary in vivo pharmacological or pharmacokineticproperties of libraries described herein that are screened include, butare not limited to, therapeutic efficacy, activity, preclinical toxicityproperties, clinical efficacy properties, clinical toxicity properties,immunogenicity, potency, and clinical safety properties.

Pharmacological or pharmacokinetic properties that may be screenedinclude, but are not limited to, cell binding affinity and cellactivity. For example, cell binding affinity assays or cell activityassays are performed to determine agonistic, antagonistic, or allostericeffects of libraries described herein. In some instances, the cellactivity assay is a cAMP assay. In some instances, libraries asdescribed herein are compared to cell binding or cell activity ofligands of GLP1R.

Libraries as described herein may be screened in cell based assays or innon-cell based assays. Examples of non-cell based assays include, butare not limited to, using viral particles, using in vitro translationproteins, and using protealiposomes with GLP1R.

Nucleic acid libraries as described herein may be screened bysequencing. In some instances, next generation sequence is used todetermine sequence enrichment of GLP1R binding variants. In someinstances, V gene distribution, J gene distribution, V gene family, CDR3counts per length, or a combination thereof is determined. In someinstances, clonal frequency, clonal accumulation, lineage accumulation,or a combination thereof is determined. In some instances, number ofsequences, sequences with VH clones, clones, clones greater than 1,clonotypes, clonotypes greater than 1, lineages, simpsons, or acombination thereof is determined. In some instances, a percentage ofnon-identical CDR3s is determined. For example, the percentage of nonidentical CDR3s is calculated as the number of non-identical CDR3s in asample divided by the total number of sequences that had a CDR3 in thesample.

Provided herein are nucleic acid libraries, wherein the nucleic acidlibraries may be expressed in a vector. Expression vectors for insertingnucleic acid libraries disclosed herein may comprise eukaryotic orprokaryotic expression vectors. Exemplary expression vectors include,without limitation, mammalian expression vectors:pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG,pSF-CMV—PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, (6His”disclosed as SEQ ID NO: 2410), pCEP4 pDEST27, pSF-CMV-Ub-KrYFP,pSF-CMV-FMDV-daGFP, pEFla-mCherry-N1 Vector, pEFla-tdTomato Vector,pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), andpSF-CMV—PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI101-AN DNA and pCambia2301; and yeast expression vectors: pTYB21 andpKLAC2, and insect vectors: pAc5.1/V5-His A and pDEST8. In someinstances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in avector to generate a construct comprising a scaffold comprisingsequences of GLP1R binding domains. In some instances, a size of theconstruct varies. In some instances, the construct comprises at least orabout 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700,1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000,4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than10000 bases. In some instances, a the construct comprises a range ofabout 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000,1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000,2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000, 3,000 to8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000,4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to 10,000, 5,000to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000,7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising GPCR binding domains, wherein the nucleic acidlibraries are expressed in a cell. In some instances, the libraries aresynthesized to express a reporter gene. Exemplary reporter genesinclude, but are not limited to, acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein,citrine fluorescent protein, orange fluorescent protein, cherryfluorescent protein, turquoise fluorescent protein, blue fluorescentprotein, horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), luciferase, and derivativesthereof. Methods to determine modulation of a reporter gene are wellknown in the art, and include, but are not limited to, fluorometricmethods (e.g. fluorescence spectroscopy, Fluorescence Activated CellSorting (FACS), fluorescence microscopy), and antibiotic resistancedetermination.

Diseases and Disorders

Provided herein are GLP1R binding libraries comprising nucleic acidsencoding for scaffolds comprising GLP1R binding domains that may havetherapeutic effects. In some instances, the GLP1R binding librariesresult in protein when translated that is used to treat a disease ordisorder. In some instances, the protein is an immunoglobulin. In someinstances, the protein is a peptidomimetic.

GLP1R libraries as described herein may comprise modulators of GLP1R. Insome instances, the modulator of GLP1R is an inhibitor. In someinstances, the modulator of GLP1R is an activator. In some instances,the GLP1R inhibitor is a GLP1R antagonist. In some instances, the GLP1Rantagonist is GLP1R-3. In some instances, GLP1R-3 comprises SEQ ID NO:2279. In some instances, GLP1R-3 comprises SEQ ID NO: 2320. Modulatorsof GLP1R, in some instances, are used for treating various diseases ordisorders.

Exemplary diseases include, but are not limited to, cancer, inflammatorydiseases or disorders, a metabolic disease or disorder, a cardiovasculardisease or disorder, a respiratory disease or disorder, pain, adigestive disease or disorder, a reproductive disease or disorder, anendocrine disease or disorder, or a neurological disease or disorder. Insome instances, the cancer is a solid cancer or a hematologic cancer. Insome instances, a modulator of GLP1R as described herein is used fortreatment of weight gain (or for inducing weight loss), treatment ofobesity, or treatment of Type II diabetes. In some instances, the GLP1Rmodulator is used for treating hypoglycemia. In some instances, theGLP1R modulator is used for treating post-bariatric hypoglycemia. Insome instances, the GLP1R modulator is used for treating severehypoglycemia. In some instances, the GLP1R modulator is used fortreating hyperinsulinism. In some instances, the GLP1R modulator is usedfor treating congenital hyperinsulinism.

In some instances, the subject is a mammal. In some instances, thesubject is a mouse, rabbit, dog, or human. Subjects treated by methodsdescribed herein may be infants, adults, or children. Pharmaceuticalcompositions comprising antibodies or antibody fragments as describedherein may be administered intravenously or subcutaneously.

Described herein are pharmaceutical compositions comprising antibodiesor antibody fragment thereof that binds GLP1R. In some embodiments, theantibody or antibody fragment thereof comprises an immunoglobulin heavychain and an immunoglobulin light chain: wherein the immunoglobulinheavy chain comprises an amino acid sequence at least about 90%, 95%,97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2303, 2304,2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321; andwherein the immunoglobulin light chain comprises an amino acid sequenceat least about 90%, 95%, 97%, 99%, or 100% identical to that set forthin SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316. In someembodiments, the antibody or antibody fragment thereof comprises animmunoglobulin heavy chain and an immunoglobulin light chain: whereinthe immunoglobulin heavy chain comprises an amino acid sequence setforth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317,2318, 2319, 2320, or 2321; and wherein the immunoglobulin light chaincomprises an amino acid sequence set forth in SEQ ID NO: 2310, 2311,2312, 2313, 2314, 2315, or 2316.

In some embodiments, the antibody or antibody fragment thereof comprisesan immunoglobulin heavy chain and an immunoglobulin light chain: whereinthe immunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2303; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2310. In some embodiments, the antibody orantibody fragment thereof comprises an immunoglobulin heavy chain and animmunoglobulin light chain: wherein the immunoglobulin heavy chaincomprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or100% identical to that set forth in SEQ ID NO: 2304; and wherein theimmunoglobulin light chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2311. In some embodiments, the antibody or antibody fragment thereofcomprises an immunoglobulin heavy chain and an immunoglobulin lightchain: wherein the immunoglobulin heavy chain comprises an amino acidsequence at least about 90%, 95%, 97%, 99%, or 100% identical to thatset forth in SEQ ID NO: 2305; and wherein the immunoglobulin light chaincomprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or100% identical to that set forth in SEQ ID NO: 2312. In someembodiments, the antibody or antibody fragment thereof comprises animmunoglobulin heavy chain and an immunoglobulin light chain: whereinthe immunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2306; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2313. In some embodiments, the antibody orantibody fragment thereof comprises an immunoglobulin heavy chain and animmunoglobulin light chain: wherein the immunoglobulin heavy chaincomprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or100% identical to that set forth in SEQ ID NO: 2307; and wherein theimmunoglobulin light chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2314. In some embodiments, the antibody or antibody fragment thereofcomprises an immunoglobulin heavy chain and an immunoglobulin lightchain: wherein the immunoglobulin heavy chain comprises an amino acidsequence at least about 90%, 95%, 97%, 99%, or 100% identical to thatset forth in SEQ ID NO: 2308; and wherein the immunoglobulin light chaincomprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or100% identical to that set forth in SEQ ID NO: 2315. In someembodiments, the antibody or antibody fragment thereof comprises animmunoglobulin heavy chain and an immunoglobulin light chain: whereinthe immunoglobulin heavy chain comprises an amino acid sequence at leastabout 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ IDNO: 2309; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90%, 95%, 97%, 99%, or 100% identical tothat set forth in SEQ ID NO: 2316.

In some instances, a pharmaceutical composition comprises an antibody orantibody fragment described herein comprising a CDR-H3 comprising asequence of any one of SEQ ID NOS: 2260-2276. In some instances, apharmaceutical composition comprises an antibody or antibody fragmentdescribed herein comprise a sequence of any one of SEQ ID NOS:2277-2295. In some instances, a pharmaceutical composition comprises anantibody or antibody fragment described herein comprise a sequence ofany one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286,2289, 2290, 2291, 2292, 2294, or 2295. In further instances, thepharmaceutical composition is used for treatment of a metabolicdisorder.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a pluralityof nucleic acids, wherein each nucleic acid encodes for a variant codonsequence compared to a reference nucleic acid sequence. In someinstances, each nucleic acid of a first nucleic acid population containsa variant at a single variant site. In some instances, the first nucleicacid population contains a plurality of variants at a single variantsite such that the first nucleic acid population contains more than onevariant at the same variant site. The first nucleic acid population maycomprise nucleic acids collectively encoding multiple codon variants atthe same variant site. The first nucleic acid population may comprisenucleic acids collectively encoding up to 19 or more codons at the sameposition. The first nucleic acid population may comprise nucleic acidscollectively encoding up to 60 variant triplets at the same position, orthe first nucleic acid population may comprise nucleic acidscollectively encoding up to 61 different triplets of codons at the sameposition. Each variant may encode for a codon that results in adifferent amino acid during translation. Table 3 provides a listing ofeach codon possible (and the representative amino acid) for a variantsite.

TABLE 2 List of codons and amino acids One Three letter letter AminoAcids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGCTGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAGPhenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine HHis CAC CAT Isoleucine I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine LLeu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AACAAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine TThr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGGTyrosine Y Tyr TAC TAT

A nucleic acid population may comprise varied nucleic acids collectivelyencoding up to 20 codon variations at multiple positions. In such cases,each nucleic acid in the population comprises variation for codons atmore than one position in the same nucleic acid. In some instances, eachnucleic acid in the population comprises variation for codons at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morecodons in a single nucleic acid. In some instances, each variant longnucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more codons in a single long nucleic acid. In someinstances, the variant nucleic acid population comprises variation forcodons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in asingle nucleic acid. In some instances, the variant nucleic acidpopulation comprises variation for codons in at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleicacid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization,parallelization, and vertical integration of the end-to-end process frompolynucleotide synthesis to gene assembly within nanowells on silicon tocreate a revolutionary synthesis platform. Devices described hereinprovide, with the same footprint as a 96-well plate, a silicon synthesisplatform is capable of increasing throughput by a factor of up to 1,000or more compared to traditional synthesis methods, with production of upto approximately 1,000,000 or more polynucleotides, or 10,000 or moregenes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomicdata has become an important factor for studies that delve into thebiological roles of various genes in both normal biology and diseasepathogenesis. At the core of this research is the central dogma ofmolecular biology and the concept of “residue-by-residue transfer ofsequential information.” Genomic information encoded in the DNA istranscribed into a message that is then translated into the protein thatis the active product within a given biological pathway.

Another exciting area of study is on the discovery, development andmanufacturing of therapeutic molecules focused on a highly-specificcellular target. High diversity DNA sequence libraries are at the coreof development pipelines for targeted therapeutics. Gene mutants areused to express proteins in a design, build, and test proteinengineering cycle that ideally culminates in an optimized gene for highexpression of a protein with high affinity for its therapeutic target.As an example, consider the binding pocket of a receptor. The ability totest all sequence permutations of all residues within the binding pocketsimultaneously will allow for a thorough exploration, increasing chancesof success. Saturation mutagenesis, in which a researcher attempts togenerate all possible mutations at a specific site within the receptor,represents one approach to this development challenge. Though costly andtime and labor-intensive, it enables each variant to be introduced intoeach position. In contrast, combinatorial mutagenesis, where a fewselected positions or short stretch of DNA may be modified extensively,generates an incomplete repertoire of variants with biasedrepresentation.

To accelerate the drug development pipeline, a library with the desiredvariants available at the intended frequency in the right positionavailable for testing—in other words, a precision library, enablesreduced costs as well as turnaround time for screening. Provided hereinare methods for synthesizing nucleic acid synthetic variant librarieswhich provide for precise introduction of each intended variant at thedesired frequency. To the end user, this translates to the ability tonot only thoroughly sample sequence space but also be able to querythese hypotheses in an efficient manner, reducing cost and screeningtime. Genome-wide editing can elucidate important pathways, librarieswhere each variant and sequence permutation can be tested for optimalfunctionality, and thousands of genes can be used to reconstruct entirepathways and genomes to re-engineer biological systems for drugdiscovery.

In a first example, a drug itself can be optimized using methodsdescribed herein. For example, to improve a specified function of anantibody, a variant polynucleotide library encoding for a portion of theantibody is designed and synthesized. A variant nucleic acid library forthe antibody can then be generated by processes described herein (e.g.,PCR mutagenesis followed by insertion into a vector). The antibody isthen expressed in a production cell line and screened for enhancedactivity. Example screens include examining modulation in bindingaffinity to an antigen, stability, or effector function (e.g., ADCC,complement, or apoptosis). Exemplary regions to optimize the antibodyinclude, without limitation, the Fc region, Fab region, variable regionof the Fab region, constant region of the Fab region, variable domain ofthe heavy chain or light chain (VII or VL), and specificcomplementarity-determining regions (CDRs) of VII or VL.

Nucleic acid libraries synthesized by methods described herein may beexpressed in various cells associated with a disease state. Cellsassociated with a disease state include cell lines, tissue samples,primary cells from a subject, cultured cells expanded from a subject, orcells in a model system. Exemplary model systems include, withoutlimitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction ortreatment of a disease state, a variant nucleic acid library describedherein is expressed in a cell associated with a disease state, or one inwhich a cell a disease state can be induced. In some instances, an agentis used to induce a disease state in cells. Exemplary tools for diseasestate induction include, without limitation, a Cre/Lox recombinationsystem, LPS inflammation induction, and streptozotocin to inducehypoglycemia. The cells associated with a disease state may be cellsfrom a model system or cultured cells, as well as cells from a subjecthaving a particular disease condition. Exemplary disease conditionsinclude a bacterial, fungal, viral, autoimmune, or proliferativedisorder (e.g., cancer). In some instances, the variant nucleic acidlibrary is expressed in the model system, cell line, or primary cellsderived from a subject, and screened for changes in at least onecellular activity. Exemplary cellular activities include, withoutlimitation, proliferation, cycle progression, cell death, adhesion,migration, reproduction, cell signaling, energy production, oxygenutilization, metabolic activity, and aging, response to free radicaldamage, or any combination thereof.

Substrates

Devices used as a surface for polynucleotide synthesis may be in theform of substrates which include, without limitation, homogenous arraysurfaces, patterned array surfaces, channels, beads, gels, and the like.Provided herein are substrates comprising a plurality of clusters,wherein each cluster comprises a plurality of loci that support theattachment and synthesis of polynucleotides. In some instances,substrates comprise a homogenous array surface. For example, thehomogenous array surface is a homogenous plate. The term “locus” as usedherein refers to a discrete region on a structure which provides supportfor polynucleotides encoding for a single predetermined sequence toextend from the surface. In some instances, a locus is on a twodimensional surface, e.g., a substantially planar surface. In someinstances, a locus is on a three-dimensional surface, e.g., a well,microwell, channel, or post. In some instances, a surface of a locuscomprises a material that is actively functionalized to attach to atleast one nucleotide for polynucleotide synthesis, or preferably, apopulation of identical nucleotides for synthesis of a population ofpolynucleotides. In some instances, polynucleotide refers to apopulation of polynucleotides encoding for the same nucleic acidsequence. In some cases, a surface of a substrate is inclusive of one ora plurality of surfaces of a substrate. The average error rates forpolynucleotides synthesized within a library described here using thesystems and methods provided are often less than 1 in 1000, less thanabout 1 in 2000, less than about 1 in 3000 or less often without errorcorrection.

Provided herein are surfaces that support the parallel synthesis of aplurality of polynucleotides having different predetermined sequences ataddressable locations on a common support. In some instances, asubstrate provides support for the synthesis of more than 50, 100, 200,400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000;20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000;1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000;4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides.In some cases, the surfaces provide support for the synthesis of morethan 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000;5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000;500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000;1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000;3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or morepolynucleotides encoding for distinct sequences. In some instances, atleast a portion of the polynucleotides have an identical sequence or areconfigured to be synthesized with an identical sequence. In someinstances, the substrate provides a surface environment for the growthof polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinctloci of a substrate, wherein each locus supports the synthesis of apopulation of polynucleotides. In some cases, each locus supports thesynthesis of a population of polynucleotides having a different sequencethan a population of polynucleotides grown on another locus. In someinstances, each polynucleotide sequence is synthesized with 1, 2, 3, 4,5, 6, 7, 8, 9 or more redundancy across different loci within the samecluster of loci on a surface for polynucleotide synthesis. In someinstances, the loci of a substrate are located within a plurality ofclusters. In some instances, a substrate comprises at least 10, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000,12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.In some instances, a substrate comprises more than 2,000; 5,000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some instances, a substratecomprises about 10,000 distinct loci. The amount of loci within a singlecluster is varied in different instances. In some cases, each clusterincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances,each cluster includes about 50-500 loci. In some instances, each clusterincludes about 100-200 loci. In some instances, each cluster includesabout 100-150 loci. In some instances, each cluster includes about 109,121, 130 or 137 loci. In some instances, each cluster includes about 19,20, 61, 64 or more loci. Alternatively or in combination, polynucleotidesynthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized ona substrate is dependent on the number of distinct loci available in thesubstrate. In some instances, the density of loci within a cluster orsurface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100,130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In somecases, a substrate comprises 10 500, 25-400, 50-500, 100-500, 150-500,10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distancebetween the centers of two adjacent loci within a cluster or surface isfrom about 10-500, from about 10-200, or from about 10-100 um. In someinstances, the distance between two centers of adjacent loci is greaterthan about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In someinstances, the distance between the centers of two adjacent loci is lessthan about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, eachlocus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is atleast or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 clusterper 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50clusters per 1 mm² or more. In some instances, a substrate comprisesfrom about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In someinstances, the distance between the centers of two adjacent clusters isat least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10,0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, eachcluster has a cross section of about 0.5 to about 2, about 0.5 to about1, or about 1 to about 2 mm. In some cases, each cluster has a crosssection of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interiorcross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 wellplate, for example between about 100 and about 200 mm by between about50 and about 150 mm. In some instances, a substrate has a diameter lessthan or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or50 mm. In some instances, the diameter of a substrate is between about25-1000, 25-800, 25 600, 25-500, 25-400, 25-300, or 25-200 mm. In someinstances, a substrate has a planar surface area of at least about 100;200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000;40,000; 50,000 mm² or more. In some instances, the thickness of asubstrate is between about 50 2000, 50-1000, 100-1000, 200-1000, or250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated fromany variety of materials suitable for the methods, compositions, andsystems described herein. In certain instances, substrate materials arefabricated to exhibit a low level of nucleotide binding. In someinstances, substrate materials are modified to generate distinctsurfaces that exhibit a high level of nucleotide binding. In someinstances, substrate materials are transparent to visible and/or UVlight. In some instances, substrate materials are sufficientlyconductive, e.g., are able to form uniform electric fields across all ora portion of a substrate. In some instances, conductive materials areconnected to an electric ground. In some instances, the substrate isheat conductive or insulated. In some instances, the materials arechemical resistant and heat resistant to support chemical or biochemicalreactions, for example polynucleotide synthesis reaction processes. Insome instances, a substrate comprises flexible materials. For flexiblematerials, materials can include, without limitation: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like. Insome instances, a substrate comprises rigid materials. For rigidmaterials, materials can include, without limitation: glass; fusesilica; silicon, plastics (for example polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). Thesubstrate, solid support or reactors can be fabricated from a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane(PDMS), and glass. The substrates/solid supports or the microstructures,reactors therein may be manufactured with a combination of materialslisted herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates have a surfacearchitecture suitable for the methods, compositions, and systemsdescribed herein. In some instances, a substrate comprises raised and/orlowered features. One benefit of having such features is an increase insurface area to support polynucleotide synthesis. In some instances, asubstrate having raised and/or lowered features is referred to as athree-dimensional substrate. In some cases, a three-dimensionalsubstrate comprises one or more channels. In some cases, one or moreloci comprise a channel. In some cases, the channels are accessible toreagent deposition via a deposition device such as a material depositiondevice. In some cases, reagents and/or fluids collect in a larger wellin fluid communication one or more channels. For example, a substratecomprises a plurality of channels corresponding to a plurality of lociwith a cluster, and the plurality of channels are in fluid communicationwith one well of the cluster. In some methods, a library ofpolynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates are configured forpolynucleotide synthesis. In some instances, the structure is configuredto allow for controlled flow and mass transfer paths for polynucleotidesynthesis on a surface. In some instances, the configuration of asubstrate allows for the controlled and even distribution of masstransfer paths, chemical exposure times, and/or wash efficacy duringpolynucleotide synthesis. In some instances, the configuration of asubstrate allows for increased sweep efficiency, for example byproviding sufficient volume for a growing polynucleotide such that theexcluded volume by the growing polynucleotide does not take up more than50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, 1%, or less of the initially available volume that is available orsuitable for growing the polynucleotide. In some instances, athree-dimensional structure allows for managed flow of fluid to allowfor the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates comprise structuressuitable for the methods, compositions, and systems described herein. Insome instances, segregation is achieved by physical structure. In someinstances, segregation is achieved by differential functionalization ofthe surface generating active and passive regions for polynucleotidesynthesis. In some instances, differential functionalization is achievedby alternating the hydrophobicity across the substrate surface, therebycreating water contact angle effects that cause beading or wetting ofthe deposited reagents. Employing larger structures can decreasesplashing and cross-contamination of distinct polynucleotide synthesislocations with reagents of the neighboring spots. In some cases, adevice, such as a material deposition device, is used to depositreagents to distinct polynucleotide synthesis locations. Substrateshaving three-dimensional features are configured in a manner that allowsfor the synthesis of a large number of polynucleotides (e.g., more thanabout 10,000) with a low error rate (e.g., less than about 1:500,1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases,a substrate comprises features with a density of about or greater thanabout 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height,and/or volume as another well of the substrate. A channel of a substratemay have the same or different width, height, and/or volume as anotherchannel of the substrate. In some instances, the diameter of a clusteror the diameter of a well comprising a cluster, or both, is betweenabout 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1,0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or0.5-2 mm. In some instances, the diameter of a cluster or well or bothis less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06,or 0.05 mm. In some instances, the diameter of a cluster or well or bothis between about 1.0 and 1.3 mm. In some instances, the diameter of acluster or well, or both is about 1.150 mm. In some instances, thediameter of a cluster or well, or both is about 0.08 mm. The diameter ofa cluster refers to clusters within a two-dimensional orthree-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000,100-1000, 200 1000, 300-1000, 400-1000, or 500-1000 um. In some cases,the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channelscorresponding to a plurality of loci within a cluster, wherein theheight or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50,or 10-50 um. In some cases, the height of a channel is less than 100,80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in asubstantially planar substrate) or both channel and locus (e.g., in athree-dimensional substrate wherein a locus corresponds to a channel) isfrom about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, forexample, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the diameter of a channel, locus, or both channel and locusis less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the distance between the center of two adjacent channels,loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200,5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface,wherein the surface comprises various surface modifications. In someinstances, the surface modifications are employed for the chemicaland/or physical alteration of a surface by an additive or subtractiveprocess to change one or more chemical and/or physical properties of asubstrate surface or a selected site or region of a substrate surface.For example, surface modifications include, without limitation, (1)changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

In some cases, the addition of a chemical layer on top of a surface(referred to as adhesion promoter) facilitates structured patterning ofloci on a surface of a substrate. Exemplary surfaces for application ofadhesion promotion include, without limitation, glass, silicon, silicondioxide and silicon nitride. In some cases, the adhesion promoter is achemical with a high surface energy. In some instances, a secondchemical layer is deposited on a surface of a substrate. In some cases,the second chemical layer has a low surface energy. In some cases,surface energy of a chemical layer coated on a surface supportslocalization of droplets on the surface. Depending on the patterningarrangement selected, the proximity of loci and/or area of fluid contactat the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto whichnucleic acids or other moieties are deposited, e.g., for polynucleotidesynthesis, are smooth or substantially planar (e.g., two-dimensional) orhave irregularities, such as raised or lowered features (e.g.,three-dimensional features). In some instances, a substrate surface ismodified with one or more different layers of compounds. Suchmodification layers of interest include, without limitation, inorganicand organic layers such as metals, metal oxides, polymers, small organicmolecules and the like.

In some instances, resolved loci of a substrate are functionalized withone or more moieties that increase and/or decrease surface energy. Insome cases, a moiety is chemically inert. In some cases, a moiety isconfigured to support a desired chemical reaction, for example, one ormore processes in a polynucleotide synthesis reaction. The surfaceenergy, or hydrophobicity, of a surface is a factor for determining theaffinity of a nucleotide to attach onto the surface. In some instances,a method for substrate functionalization comprises: (a) providing asubstrate having a surface that comprises silicon dioxide; and (b)silanizing the surface using, a suitable silanizing agent describedherein or otherwise known in the art, for example, an organofunctionalalkoxysilane molecule. Methods and functionalizing agents are describedin U.S. Pat. No. 5,474,796, which is herein incorporated by reference inits entirety.

In some instances, a substrate surface is functionalized by contact witha derivatizing composition that contains a mixture of silanes, underreaction conditions effective to couple the silanes to the substratesurface, typically via reactive hydrophilic moieties present on thesubstrate surface. Silanization generally covers a surface throughself-assembly with organofunctional alkoxysilane molecules. A variety ofsiloxane functionalizing reagents can further be used as currently knownin the art, e.g., for lowering or increasing surface energy. Theorganofunctional alkoxysilanes are classified according to their organicfunctions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis mayinclude processes involving phosphoramidite chemistry. In someinstances, polynucleotide synthesis comprises coupling a base withphosphoramidite. Polynucleotide synthesis may comprise coupling a baseby deposition of phosphoramidite under coupling conditions, wherein thesame base is optionally deposited with phosphoramidite more than once,i.e., double coupling. Polynucleotide synthesis may comprise capping ofunreacted sites. In some instances, capping is optional. Polynucleotidesynthesis may also comprise oxidation or an oxidation step or oxidationsteps. Polynucleotide synthesis may comprise deblocking, detritylation,and sulfurization. In some instances, polynucleotide synthesis compriseseither oxidation or sulfurization. In some instances, between one oreach step during a polynucleotide synthesis reaction, the device iswashed, for example, using tetrazole or acetonitrile. Time frames forany one step in a phosphoramidite synthesis method may be less thanabout 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise asubsequent addition of a phosphoramidite building block (e.g.,nucleoside phosphoramidite) to a growing polynucleotide chain for theformation of a phosphite triester linkage. Phosphoramiditepolynucleotide synthesis proceeds in the 3′ to 5′ direction.Phosphoramidite polynucleotide synthesis allows for the controlledaddition of one nucleotide to a growing nucleic acid chain per synthesiscycle. In some instances, each synthesis cycle comprises a couplingstep. Phosphoramidite coupling involves the formation of a phosphitetriester linkage between an activated nucleoside phosphoramidite and anucleoside bound to the substrate, for example, via a linker. In someinstances, the nucleoside phosphoramidite is provided to the deviceactivated. In some instances, the nucleoside phosphoramidite is providedto the device with an activator. In some instances, nucleosidephosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition of a nucleosidephosphoramidite, the device is optionally washed. In some instances, thecoupling step is repeated one or more additional times, optionally witha wash step between nucleoside phosphoramidite additions to thesubstrate. In some instances, a polynucleotide synthesis method usedherein comprises 1, 2, 3 or more sequential coupling steps. Prior tocoupling, in many cases, the nucleoside bound to the device isde-protected by removal of a protecting group, where the protectinggroup functions to prevent polymerization. A common protecting group is4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step is usefulto block unreacted substrate-bound 5′-OH groups after coupling fromfurther chain elongation, preventing the formation of polynucleotideswith internal base deletions. Further, phosphoramidites activated with1H-tetrazole may react, to a small extent, with the O6 position ofguanosine. Without being bound by theory, upon oxidation with I2/water,this side product, possibly via O6-N7 migration, may undergodepurination. The apurinic sites may end up being cleaved in the courseof the final deprotection of the polynucleotide thus reducing the yieldof the full-length product. The O6 modifications may be removed bytreatment with the capping reagent prior to oxidation with 12/water. Insome instances, inclusion of a capping step during polynucleotidesynthesis decreases the error rate as compared to synthesis withoutcapping. As an example, the capping step comprises treating thesubstrate-bound polynucleotide with a mixture of acetic anhydride and1-methylimidazole. Following a capping step, the device is optionallywashed.

In some instances, following addition of a nucleoside phosphoramidite,and optionally after capping and one or more wash steps, the devicebound growing nucleic acid is oxidized. The oxidation step comprises thephosphite triester is oxidized into a tetracoordinated phosphatetriester, a protected precursor of the naturally occurring phosphatediester internucleoside linkage. In some instances, oxidation of thegrowing polynucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base (e.g., pyridine, lutidine,collidine). Oxidation may be carried out under anhydrous conditionsusing, e.g. tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for device drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, the deviceand growing polynucleotide is optionally washed. In some instances, thestep of oxidation is substituted with a sulfurization step to obtainpolynucleotide phosphorothioates, wherein any capping steps can beperformed after the sulfurization. Many reagents are capable of theefficient sulfur transfer, including but not limited to3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occurthrough coupling, the protected 5′ end of the device bound growingpolynucleotide is removed so that the primary hydroxyl group is reactivewith a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound polynucleotide and thus reduces theyield of the desired full-length product. Methods and compositions ofthe disclosure described herein provide for controlled deblockingconditions limiting undesired depurination reactions. In some instances,the device bound polynucleotide is washed after deblocking. In someinstances, efficient washing after deblocking contributes to synthesizedpolynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve aniterating sequence of the following steps: application of a protectedmonomer to an actively functionalized surface (e.g., locus) to link witheither the activated surface, a linker or with a previously deprotectedmonomer; deprotection of the applied monomer so that it is reactive witha subsequently applied protected monomer; and application of anotherprotected monomer for linking. One or more intermediate steps includeoxidation or sulfurization. In some instances, one or more wash stepsprecede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise aseries of chemical steps. In some instances, one or more steps of asynthesis method involve reagent cycling, where one or more steps of themethod comprise application to the device of a reagent useful for thestep. For example, reagents are cycled by a series of liquid depositionand vacuum drying steps. For substrates comprising three-dimensionalfeatures such as wells, microwells, channels and the like, reagents areoptionally passed through one or more regions of the device via thewells and/or channels.

Methods and systems described herein relate to polynucleotide synthesisdevices for the synthesis of polynucleotides. The synthesis may be inparallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or morepolynucleotides can be synthesized in parallel. The total numberpolynucleotides that may be synthesized in parallel may be from2-100000, 3-50000, 4 10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700,11-650, 12-600, 13-550, 14-500, 15-450, 16 400, 17-350, 18-300, 19-250,20-200, 21-150, 22-100, 23-50, 24-45, 25-40, 30-35. Those of skill inthe art appreciate that the total number of polynucleotides synthesizedin parallel may fall within any range bound by any of these values, forexample 25-100. The total number of polynucleotides synthesized inparallel may fall within any range defined by any of the values servingas endpoints of the range. Total molar mass of polynucleotidessynthesized within the device or the molar mass of each of thepolynucleotides may be at least or at least about 10, 20, 30, 40, 50,100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The lengthof each of the polynucleotides or average length of the polynucleotideswithin the device may be at least or about at least 10, 15, 20, 25, 30,35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. Thelength of each of the polynucleotides or average length of thepolynucleotides within the device may be at most or about at most 500,400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10 nucleotides, or less. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100,15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciatethat the length of each of the polynucleotides or average length of thepolynucleotides within the device may fall within any range bound by anyof these values, for example 100-300. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall within any range defined by any of the values serving asendpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allowfor synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175,200 nucleotides per hour, or more are synthesized. Nucleotides includeadenine, guanine, thymine, cytosine, uridine building blocks, oranalogs/modified versions thereof. In some instances, libraries ofpolynucleotides are synthesized in parallel on substrate. For example, adevice comprising about or at least about 100; 1,000; 10,000; 30,000;75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or5,000,000 resolved loci is able to support the synthesis of at least thesame number of distinct polynucleotides, wherein polynucleotide encodinga distinct sequence is synthesized on a resolved locus. In someinstances, a library of polynucleotides is synthesized on a device withlow error rates described herein in less than about three months, twomonths, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acidsassembled from a polynucleotide library synthesized with low error rateusing the substrates and methods described herein are prepared in lessthan about three months, two months, one month, three weeks, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of alibrary of nucleic acids comprising variant nucleic acids differing at aplurality of codon sites. In some instances, a nucleic acid may have 1site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may beadjacent. In some instances, the one or more sites of variant codonsites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variantcodon sites, wherein all the variant codon sites are adjacent to oneanother, forming a stretch of variant codon sites. In some instances, anucleic acid may comprise multiple sites of variant codon sites, whereinnone the variant codon sites are adjacent to one another. In someinstances, a nucleic acid may comprise multiple sites of variant codonsites, wherein some the variant codon sites are adjacent to one another,forming a stretch of variant codon sites, and some of the variant codonsites are not adjacent to one another.

Referring to the Figures, FIG. 3 illustrates an exemplary processworkflow for synthesis of nucleic acids (e.g., genes) from shorternucleic acids. The workflow is divided generally into phases: (1) denovo synthesis of a single stranded nucleic acid library, (2) joiningnucleic acids to form larger fragments, (3) error correction, (4)quality control, and (5) shipment. Prior to de novo synthesis, anintended nucleic acid sequence or group of nucleic acid sequences ispreselected. For example, a group of genes is preselected forgeneration.

Once large nucleic acids for generation are selected, a predeterminedlibrary of nucleic acids is designed for de novo synthesis. Varioussuitable methods are known for generating high density polynucleotidearrays. In the workflow example, a device surface layer is provided. Inthe example, chemistry of the surface is altered in order to improve thepolynucleotide synthesis process. Areas of low surface energy aregenerated to repel liquid while areas of high surface energy aregenerated to attract liquids. The surface itself may be in the form of aplanar surface or contain variations in shape, such as protrusions ormicrowells which increase surface area. In the workflow example, highsurface energy molecules selected serve a dual function of supportingDNA chemistry, as disclosed in International Patent ApplicationPublication WO/2015/021080, which is herein incorporated by reference inits entirety.

In situ preparation of polynucleotide arrays is generated on a solidsupport and utilizes single nucleotide extension process to extendmultiple oligomers in parallel. A deposition device, such as a materialdeposition device, is designed to release reagents in a step wisefashion such that multiple polynucleotides extend, in parallel, oneresidue at a time to generate oligomers with a predetermined nucleicacid sequence 302. In some instances, polynucleotides are cleaved fromthe surface at this stage. Cleavage includes gas cleavage, e.g., withammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber.In this exemplary workflow, the reaction chamber (also referred to as“nanoreactor”) is a silicon coated well, containing PCR reagents andlowered onto the polynucleotide library 303. Prior to or after thesealing 304 of the polynucleotides, a reagent is added to release thepolynucleotides from the substrate. In the exemplary workflow, thepolynucleotides are released subsequent to sealing of the nanoreactor305. Once released, fragments of single stranded polynucleotideshybridize in order to span an entire long range sequence of DNA. Partialhybridization 305 is possible because each synthesized polynucleotide isdesigned to have a small portion overlapping with at least one otherpolynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerasecycles, the polynucleotides anneal to complementary fragments and gapsare filled in by a polymerase. Each cycle increases the length ofvarious fragments randomly depending on which polynucleotides find eachother. Complementarity amongst the fragments allows for forming acomplete large span of double stranded DNA 306.

After PCA is complete, the nanoreactor is separated from the device 307and positioned for interaction with a device having primers for PCR 308.After sealing, the nanoreactor is subject to PCR 309 and the largernucleic acids are amplified. After PCR 310, the nanochamber is opened311, error correction reagents are added 312, the chamber is sealed 313and an error correction reaction occurs to remove mismatched base pairsand/or strands with poor complementarity from the double stranded PCRamplification products 314. The nanoreactor is opened and separated 315.Error corrected product is next subject to additional processing steps,such as PCR and molecular bar coding, and then packaged 322 for shipment323.

In some instances, quality control measures are taken. After errorcorrection, quality control steps include for example interaction with awafer having sequencing primers for amplification of the error correctedproduct 316, sealing the wafer to a chamber containing error correctedamplification product 317, and performing an additional round ofamplification 318. The nanoreactor is opened 319 and the products arepooled 320 and sequenced 321. After an acceptable quality controldetermination is made, the packaged product 322 is approved for shipment323.

In some instances, a nucleic acid generated by a workflow such as thatin FIG. 3 is subject to mutagenesis using overlapping primers disclosedherein. In some instances, a library of primers are generated by in situpreparation on a solid support and utilize single nucleotide extensionprocess to extend multiple oligomers in parallel. A deposition device,such as a material deposition device, is designed to release reagents ina step wise fashion such that multiple polynucleotides extend, inparallel, one residue at a time to generate oligomers with apredetermined nucleic acid sequence 302.

Computer Systems

Any of the systems described herein, may be operably linked to acomputer and may be automated through a computer either locally orremotely. In various instances, the methods and systems of thedisclosure may further comprise software programs on computer systemsand use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of thedisclosure. The computer systems may be programmed to interface betweenthe user specified base sequence and the position of a materialdeposition device to deliver the correct reagents to specified regionsof the substrate.

The computer system 400 illustrated in FIG. 4 may be understood as alogical apparatus that can read instructions from media 411 and/or anetwork port 405, which can optionally be connected to server 409 havingfixed media 412. The system, such as shown in FIG. 4 can include a CPU401, disk drives 403, optional input devices such as keyboard 415 and/ormouse 416 and optional monitor 407. Data communication can be achievedthrough the indicated communication medium to a server at a local or aremote location. The communication medium can include any means oftransmitting and/or receiving data. For example, the communicationmedium can be a network connection, a wireless connection or an internetconnection. Such a connection can provide for communication over theWorld Wide Web. It is envisioned that data relating to the presentdisclosure can be transmitted over such networks or connections forreception and/or review by a party 422 as illustrated in FIG. 4.

FIG. 5 is a block diagram illustrating a first example architecture of acomputer system 500 that can be used in connection with exampleinstances of the present disclosure. As depicted in FIG. 5, the examplecomputer system can include a processor 502 for processing instructions.Non-limiting examples of processors include: Intel Xeon™ processor, AMDOpteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor,ARM Cortex-A8 Samsung S5PC100TM processor, ARM Cortex-A8 Apple A4™processor, Marvell PXA 930™ processor, or a functionally-equivalentprocessor. Multiple threads of execution can be used for parallelprocessing. In some instances, multiple processors or processors withmultiple cores can also be used, whether in a single computer system, ina cluster, or distributed across systems over a network comprising aplurality of computers, cell phones, and/or personal data assistantdevices.

As illustrated in FIG. 5, a high speed cache 504 can be connected to, orincorporated in, the processor 502 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used bythe processor 502. The processor 502 is connected to a north bridge 506by a processor bus 508. The north bridge 506 is connected to randomaccess memory (RAM) 510 by a memory bus 512 and manages access to theRAM 510 by the processor 502. The north bridge 506 is also connected toa south bridge 514 by a chipset bus 516. The south bridge 514 is, inturn, connected to a peripheral bus 518. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 518. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip. In someinstances, system 500 can include an accelerator card 522 attached tothe peripheral bus 518. The accelerator can include field programmablegate arrays (FPGAs) or other hardware for accelerating certainprocessing. For example, an accelerator can be used for adaptive datarestructuring or to evaluate algebraic expressions used in extended setprocessing.

Software and data are stored in external storage 524 and can be loadedinto RAM 510 and/or cache 504 for use by the processor. The system 500includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample instances of the present disclosure. In this example, system 500also includes network interface cards (NICs) 520 and 521 connected tothe peripheral bus for providing network interfaces to external storage,such as Network Attached Storage (NAS) and other computer systems thatcan be used for distributed parallel processing.

FIG. 6 is a diagram showing a network 600 with a plurality of computersystems 602 a, and 602 b, a plurality of cell phones and personal dataassistants 602 c, and Network Attached Storage (NAS) 604 a, and 604 b.In example instances, systems 602 a, 602 b, and 602 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 604 a and 604 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 602 a, and 602 b, and cell phone and personal dataassistant systems 602 c. Computer systems 602 a, and 602 b, and cellphone and personal data assistant systems 602 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 604 a and 604 b. FIG. 6 illustrates anexample only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various instances of thepresent disclosure. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface. In some example instances, processors can maintainseparate memory spaces and transmit data through network interfaces,back plane or other connectors for parallel processing by otherprocessors. In other instances, some or all of the processors can use ashared virtual address memory space.

FIG. 7 is a block diagram of a multiprocessor computer system 700 usinga shared virtual address memory space in accordance with an exampleinstance. The system includes a plurality of processors 702 a-f that canaccess a shared memory subsystem 704. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)706 a-f in the memory subsystem 704. Each MAP 706 a-f can comprise amemory 708 a-f and one or more field programmable gate arrays (FPGAs)710 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 710a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample instances. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 708a-f, allowing it to execute tasks independently of, and asynchronouslyfrom the respective microprocessor 702 a-f. In this configuration, a MAPcan feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleinstances, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some instances, allor part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example instances, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other instances, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 5, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 522 illustrated in FIG. 5.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of thedisclosure. Changes therein and other uses which are encompassed withinthe spirit of the disclosure as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of polynucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with Nz. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200× spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A descum process was used to remove residualresist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 wattsfor 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min, and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withNz. The functionalized surface was activated to serve as a support forpolynucleotide synthesis.

Example 2: Synthesis of a 50-Mer Sequence on an OligonucleotideSynthesis Device

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to a flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used tosynthesize an exemplary polynucleotide of 50 bp (“50-merpolynucleotide”) using polynucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO.: 2.5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT ## TTTTTTT TTT3′(SEQ ID NO.: 2), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer.

TABLE 3 Synthesis protocols General DNA Synthesis Table 3 Process NameProcess Step Time (sec) WASH (Acetonitrile Wash Acetonitrile SystemFlush 4 Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Incubate for 25sec 25 WASH (AcetonitrileWash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION ActivatorManifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 ActivatorFlow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25sec 25WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrileto Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPING(CapA + B, CapA + B to Flowcell 15 1:1, Flow) WASH (Acetonitrile WashAcetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15Acetonitrile System Flush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 SystemFlush 4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock toFlowcell 36 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile toFlowcell 18 N2 System Flush 4.13 Acetonitrile System Flush 4.13Acetonitrile to Flowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flowrestrictor). The time to completely push out Oxidizer was observed, thetiming for chemical flow times was adjusted accordingly and an extra ACNwash was introduced between different chemicals. After polynucleotidesynthesis, the chip was deprotected in gaseous ammonia overnight at 75psi. Five drops of water were applied to the surface to recoverpolynucleotides. The recovered polynucleotides were then analyzed on aBioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an OligonucleotideSynthesis Device

The same process as described in Example 2 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer polynucleotide(“100-mer polynucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT ## TTTTTTTTTT3′, where #denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO.: 3) on two different silicon chips, the first oneuniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the polynucleotides extracted from thesurface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGGTTCTCATCATC3; SEQ ID NO.: 4) and a reverse(5′CGGGATCCTTATCGTCATCG3; SEQ ID NO.: 5) primer in a 50 uL PCR mix (25uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL polynucleotide extracted from the surface, and water up to50 uL) using the following thermalcycling program: 98° C., 30 sec 98°C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles 72° C., 2min

The PCR products were also run on a BioAnalyzer, demonstrating sharppeaks at the 100-mer position. Next, the PCR amplified samples werecloned, and Sanger sequenced. Table 4 summarizes the results from theSanger sequencing for samples taken from spots 1-5 from chip 1 and forsamples taken from spots 6-10 from chip 2.

TABLE 4 Sequencing results Spot Error rate Cycle efficiency  1 1/763 bp99.87%  2 1/824 bp 99.88%  3 1/780 bp 99.87%  4 1/429 bp 99.77%  51/1525 bp  99.93%  6 1/1615 bp  99.94%  7 1/531 bp 99.81%  8 1/1769 bp 99.94%  9 1/854 bp 99.88% 10 1/1451 bp  99.93%

Thus, the high quality and uniformity of the synthesized polynucleotideswere repeated on two chips with different surface chemistries. Overall,89% of the 100-mers that were sequenced were perfect sequences with noerrors, corresponding to 233 out of 262.

Table 5 summarizes error characteristics for the sequences obtained fromthe polynucleotide samples from spots 1-10.

TABLE 5 Error characteristics Sample ID/ Spot no. OSA_0046/1 OSA_0047/2OSA_0048/3 OSA_0049/4 OSA_0050/5 OSA_0051/6 Total Sequences 32 32 32 3232 32 Sequencing 25 of 28 27 of 27 26 of 30 21 of 23 25 of 26 29 of 30Quality Oligo Quality 23 of 25 25 of 27 22 of 26 18 of 21 24 of 25 25 of29 ROI Match Count 2500 2698 2561 2122 2499 2666 ROI Mutation 2 2 1 3 10 ROI Multi Base 0 0 0 0 0 0 Deletion ROI Small 1 0 0 0 0 0 InsertionROI Single 0 0 0 0 0 0 Base Deletion Large Deletion 0 0 1 0 0 1 CountMutation: G > A 2 2 1 2 1 0 Mutation: T > C 0 0 0 1 0 0 ROI Error Count3 2 2 3 1 1 ROI Error Rate Err: ~1 in Err: ~1 in Err: ~1 in Err: ~1 inErr: ~1 in Err: ~1 in 834 1350 1282 708 2500 2667 ROI Minus Primer MPErr: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 ErrorRate in 763 in 824 in 780 in 429 in 1525 in 1615 Sample ID/ Spot no.OSA_0052/7 OSA_0053/8 OSA_0054/9 OSA_0055/10 Total Sequences 32 32 32 32Sequencing Quality 27 of 31 29 of 31 28 of 29 25 of 28 Oligo Quality 22of 27 28 of 29 26 of 28 20 of 25 ROI Match Count 2625 2899 2798 2348 ROIMutation 2 1 2 1 ROI Multi Base 0 0 0 0 Deletion ROI Small 0 0 0 0Insertion ROI Single Base 0 0 0 0 Deletion Large Deletion 1 0 0 0 CountMutation: G > A 2 1 2 1 Mutation: T > C 0 0 0 0 ROI Error Count 3 1 2 1ROI Error Rate Err: ~1 in 876 Err: ~1 in 2900 Err: ~1 in 1400 Err: ~1 in2349 ROI Minus Primer MP Err: ~1 in MP Err: ~1 in MP Err: ~1 in MP Err:~1 in Error Rate 531 1769 854 1451

Example 4: Design of GLP1R Binding Domains Based on Peptide LigandInteractions

GLP1R binding domains were designed based on interaction surfacesbetween peptide ligands that interact with GLP1R. Motif variants weregenerated based on the interaction surface of the peptides with the ECDas well as with the N-terminal GLP1R ligand interaction surface. Thiswas done using structural modeling. Exemplary motif variants werecreated based on glucagon like peptide's interaction with GLP1R as seenin Table 6. The motif variant sequences were generated using thefollowing sequence from glucagon like peptide:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. (SEQ ID NO: 6)

TABLE 6 Variant amino acid sequences for glucagon like peptide SEQ IDNO. Variant Amino Acid Sequence  7 1 sggggsggggsggggHAEGTFTSDVSSYLEGQAAKEFIAWLV  8 2 sggggsggggsggggAEGTFTSDVSSYLEGQAAK EFIAWLV  9 3sggggsggggsggggEGTFTSDVSSYLEGQAAKE FIAWLV 10 4sggggsggggsggggGTFTSDVSSYLEGQAAKEF IAWLV 11 5sggggsggggsggggTFTSDVSSYLEGQAAKEFI AWLV 12 6sggggsggggsggggFTSDVSSYLEGQAAKEFIA WLV 13 7sggggsggggsggggTSDVSSYLEGQAAKEFIAW LV 14 8sggggsggggsggggSDVSSYLEGQAAKEFIAWL V 15 9sggggsggggsggggDVSSYLEGQAAKEFIAWLV

Example 5: Design of Antibody Scaffolds

To generate scaffolds, structural analysis, repertoire sequencinganalysis of the heavy chain, and specific analysis of heterodimerhigh-throughput sequencing datasets were performed. Each heavy chain wasassociated with each light chain scaffold. Each heavy chain scaffold wasassigned 5 different long CDR-H3 loop options. Each light chain scaffoldwas assigned 5 different L3 scaffolds. The heavy chain CDR-H3 stems werechosen from the frequently observed long H3 loop stems (10 amino acidson the N-terminus and the C-terminus) found both across individuals andacross V-gene segments. The light chain scaffold L3s were chosen fromheterodimers comprising long H3s. Direct heterodimers based oninformation from the Protein Data Bank (PDB) and deep sequencingdatasets were used in which CDR H1, H2, L1, L2, L3, and CDR-H3 stemswere fixed. The various scaffolds were then formatted for display onphage to assess for expression.

Structural Analysis

About 2,017 antibody structures were analyzed from which 22 structureswith long CDR-H3s of at least 25 amino acids in length were observed.The heavy chains included the following: IGHV1-69, IGHV3-30, IGHV4-49,and IGHV3-21. The light chains identified included the following:IGLV3-21, IGKV3-11, IGKV2-28, IGKV1-5, IGLV1-51, IGLV1-44, and IGKV1-13.In the analysis, four heterodimer combinations were observed multipletimes including: IGHV4-59/61-IGLV3-21, IGHV3-21-IGKV2-28,IGHV1-69-IGKV3-11, and IGHV1-69-IGKV1-5. An analysis of sequences andstructures identified intra-CDR-H3 disulfide bonds in a few structureswith packing of bulky side chains such as tyrosine in the stem providingsupport for long H₃ stability. Secondary structures includingbeta-turn-beta sheets and a “hammerhead” subdomain were also observed.

Repertoire Analysis

A repertoire analysis was performed on 1,083,875 IgM+/CD27-naive B cellreceptor (BCR) sequences and 1,433,011 CD27+ sequences obtained byunbiased 5′RACE from 12 healthy controls. The 12 healthy controlscomprised equal numbers of male and female and were made up of 4Caucasian, 4 Asian, and 4 Hispanic individuals. The repertoire analysisdemonstrated that less than 1% of the human repertoire comprises BCRswith CDR-H3s longer than 21 amino acids. A V-gene bias was observed inthe long CDR3 subrepertoire, with IGHV1-69, IGHV4-34, IGHV1-18, andIGHV1-8 showing preferential enrichment in BCRs with long H3 loops. Abias against long loops was observed for IGHV3-23, IGHV4-59/61,IGHVS-51, IGHV3-48, IGHV3-53/66, IGHV3-15, IGHV3-74, IGHV3-73, IGHV3-72,and IGHV2-70. The IGHV4-34 scaffold was demonstrated to be autoreactiveand had a short half-life.

Viable N-terminal and C-terminal CDR-H3 scaffold variation for longloops were also designed based on the 5′RACE reference repertoire. About81,065 CDR-H3s of amino acid length 22 amino acids or greater wereobserved. By comparing across V-gene scaffolds, scaffold-specific H₃stem variation was avoided as to allow the scaffold diversity to becloned into multiple scaffold references.

Heterodimer Analysis

Heterodimer analysis was performed on scaffolds and variant sequencesand lengths of the scaffolds were assayed.

Structural Analysis

Structural analysis was performed using GPCR scaffolds of variantsequences and lengths were assayed.

Example 6: Generation of GPCR Antibody Libraries

Based on GPCR-ligand interaction surfaces and scaffold arrangements,libraries were designed and de novo synthesized. See Example 4. 10variant sequences were designed for the variable domain, heavy chain,237 variant sequences were designed for the heavy chain complementaritydetermining region 3, and 44 variant sequences were designed for thevariable domain, light chain. The fragments were synthesized as threefragments following similar methods as described in Examples 1-3.

Following de novo synthesis, 10 variant sequences were generated for thevariable domain, heavy chain, 236 variant sequences were generated forthe heavy chain complementarity determining region 3, and 43 variantsequences were designed for a region comprising the variable domain,light chain and CDR-L3 and of which 9 variants for variable domain,light chain were designed. This resulted in a library with about 10⁵diversity (10×236×43). This was confirmed using next generationsequencing (NGS) with 16 million reads. The normalized sequencing readsfor each of the 10 variants for the variable domain, heavy chain wasabout 1 (data not shown). The normalized sequencing reads for each ofthe 43 variants for the variable domain, light chain was about 1 (datanot shown). The normalized sequencing reads for 236 variant sequencesfor the heavy chain complementarily determining region 3 were about 1(data not shown).

The various light and heavy chains were then tested for expression andprotein folding. The 10 variant sequences for variable domain, heavychain included the following: IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21,IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, and IGHV4-59/61.Of the 10 variant sequences, IGHV1-18, IGHV1-69, and IGHV3-30/33rnexhibited improved characteristics such as improved thermostability. 9variant sequences for variable domain, light chain included thefollowing: IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20,IGKV4-1, IGLV1-51, and IGLV2-14. Of the 9 variant sequences, IGKV1-39,IGKV3-15, IGLV1-51, and IGLV2-14 exhibited improved characteristics suchas improved thermostability.

Example 7: Expression of GPCR Antibody Libraries in HEK293 Cells

Following generation of GPCR antibody libraries, about 47 GPCRs wereselected for screening. GPCR constructs about 1.8 kb to about 4.5 kb insize were designed in a pCDNA3.1 vector. The GPCR constructs were thensynthesized following similar methods as described in Examples 2-4including hierarchal assembly. Of the 47 GPCR constructs, 46 GPCRconstructs were synthesized.

The synthesized GPCR constructs were transfected in HEK293 and assayedfor expression using immunofluorescence. HEK293 cells were transfectedwith the GPCR constructs comprising an N-terminally hemagglutinin(HA)-tagged human Y₁ receptor. Following 24-48 hours of transfection,cells were washed with phosphate buffered saline (PBS) and fixed with 4%paraformaldehyde. Cells were stained using fluorescent primary antibodydirected towards the HA tag or secondary antibodies comprising afluorophore and DAPI to visualize the nuclei in blue. Human Y₁ receptorwas visualized on the cell surface in non-permeabilized cells and on thecell surface and intracellularly in permeabilized cells.

GPCR constructs were also visualized by designing GPCR constructscomprising auto-fluorescent proteins. Human Y₁ receptor comprised EYFPfused to its C-terminus, and human Y₅ receptor comprised ECFP fused toits C-terminus. HEK293 cells were transfected with human Y₁ receptor orco-transfected with human Y₁ receptor and human Y₅ receptor. Followingtransfection cells were washed and fixed with 4% paraformaldehyde. Cellswere stained with DAPI. Localization of human Y₁ receptor and human Y₅receptor were visualized by fluorescence microscopy.

Example 8: Design of Immunoglobulin Library

An immunoglobulin scaffold library was designed for placement of GPCRbinding domains and for improving stability for a range of GPCR bindingdomain encoding sequences. The immunoglobulin scaffold included a VHdomain attached with a VL domain with a linker. Variant nucleic acidsequences were generated for the framework elements and CDR elements ofthe VH domain and VL domain. The structure of the design is shown inFIG. 8A. A full domain architecture is shown in FIG. 8B. Sequences forthe leader, linker, and pIII are listed in Table 7.

TABLE 7 Nucleotide sequences SEQ ID NO Domain Sequence 16 LeaderGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAG CCGGCCATGGCC 17 LinkerGCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGC TCTGGCGGTGGCGGATCGCATGCATCC 18 pIIICGCGCGGCCGCTGGAAGCGGCTCCCACCATCAC CATCACCAT

The VL domains that were designed include IGKV1-39, IGKV3-15, IGLV1-51,and IGLV2-14. Each of four VL domains were assembled with theirrespective invariant four framework elements (FW1, FW2, FW3, FW4) andvariable 3 CDR (L1, L2, L3) elements. For IGKV1-39, there was 490variants designed for L1, 420 variants designed for L2, and 824 variantsdesigned for L3 resulting in a diversity of 1.7×10⁸ (490*420*824). ForIGKV3-15, there was 490 variants designed for L1, 265 variants designedfor L2, and 907 variants designed for L3 resulting in a diversity of1.2×10⁸ (490*265*907). For IGLV 1-51, there was 184 variants designedfor L1, 151 variants designed for L2, and 824 variants designed for L3resulting in a diversity of 2.3×10⁷ (184*151*824). IGLV2-14, 967variants designed for L1, 535 variants designed for L2, and 922 variantsdesigned for L3 resulting in a diversity of 4.8 10⁸ (967*535*922). Table8 lists the amino acid sequences and nucleotide sequences for the fourframework elements (FW1, FW2, FW3, FW4) for IGLV 1-51. Table 9 lists thevariable 3 CDR (L1, L2, L3) elements for IGLV 1-51. Variant amino acidsequences and nucleotide sequences for the four framework elements (FW1,FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements were alsodesigned for IGKV1-39, IGKV3-15, and IGLV2-14.

TABLE 8 Sequences for IGLV1-51 framework elements SEQ SEQ ID Amino AcidID Element NO Sequence NO Nucleotide Sequence IGLV1-51 FW1 19QSVLTQPPSVS 20 CAGTCTGTGTTGACGCAGCCG AAPGQKVTISC CCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCC TGC FW2 21 WYQQLPGTAPK 22 TGGTATCAGCAGCTCCCAGGALLIY ACAGCCCCCAAACTCCTCATT TAT FW3 23 GIPDRFSGSKS 24GGGATTCCTGACCGATTCTCT GGCTCCAAGTCTGGCACGTCA GTSATLGITGLGCCACCCTGGGCATCACCGGA QTGDEADYY CTCCAGACTGGGGACGAGGCC GATTATTAC FW4 25GGGTKLTVL 26 GGCGGAGGGACCAAGCTGACC GTCCTA

TABLE 9 Sequences for IGLV1-51 CDR elements SEQ SEQ ID Amino Acid ID NOSequence NO Nucleotide Sequence IGLV1-51-L1   27 SGSSSNIGSNHVS  210TCTGGAAGCAGCTCCAACATTGGGAGTAATCATGTATCC   28 SGSSSNIGNNYLS  211TCTGGAAGCAGCTCCAACATTGGGAATAATTATCTATCC   29 SGSSSNIANNYVS  212TCTGGAAGCAGCTCCAACATTGCGAATAATTATGTATCC   30 SGSSPNIGNNYVS  213TCTGGAAGCAGCCCCAACATTGGGAATAATTATGTATCG   31 SGSRSNIGSNYVS  214TCTGGAAGCAGATCCAATATTGGGAGTAATTATGTTTCG   32 SGSSSNVGDNYVS  215TCTGGAAGCAGCTCCAACGTTGGCGATAATTATGTTTCC   33 SGSSSNIGIQYVS  216TCTGGAAGCAGCTCCAACATTGGGATTCAATATGTATCC   34 SGSSSNVGNNFVS  217TCTGGAAGCAGCTCCAATGTTGGTAACAATTTTGTCTCC   35 SGSASNIGNNYVS  218TCTGGAAGCGCCTCCAACATTGGGAATAATTATGTATCC   36 SGSGSNIGNNDVS  219TCTGGAAGCGGCTCCAATATTGGGAATAATGATGTGTCC   37 SGSISNIGNNYVS  220TCTGGAAGCATCTCCAACATTGGTAATAATTATGTATCC   38 SGSISNIGKNYVS  221TCTGGAAGCATCTCCAACATTGGGAAAAATTATGTGTCG   39 SGSSSNIGHNYVS  222TCTGGAAGCAGCTCCAACATTGGGCATAATTATGTATCG   40 PGSSSNIGNNYVS  223CCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC   41 SGSTSNIGIHYVS  224TCTGGAAGCACCTCCAACATTGGAATTCATTATGTATCC   42 SGSSSNIGSHYVS  225TCTGGAAGCAGCTCCAACATTGGCAGTCATTATGTTTCC   43 SGSSSNIGNEYVS  226TCCGGAAGCAGCTCCAACATTGGAAATGAATATGTATCC   44 SGSTSNIGNNYIS  227TCTGGAAGCACCTCCAACATTGGAAATAATTATATATCG   45 SGSSSNIGNHFVS  228TCTGGAAGCAGCTCCAATATTGGGAATCATTTTGTATCG   46 SGSSSNIGNNYVA  229TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTGGCC   47 SGSSSNIGSYYVS  230TCTGGAAGCAGCTCCAACATTGGAAGTTATTATGTATCC   48 SGSGFNIGNNYVS  231TCTGGAAGTGGTTTCAACATTGGGAATAATTATGTCTCT   49 SGSTSNIGNNYVS  232TCTGGAAGCACCTCCAACATTGGGAATAATTATGTGTCC   50 SGSSSDIGNNYVS  233TCTGGAAGCAGCTCCGACATTGGCAATAATTATGTATCC   51 SGSSSNIGNNVVS  234TCTGGAAGCAGCTCCAACATTGGGAATAATGTTGTATCC   52 SGSKSNIGKNYVS  235TCTGGAAGCAAGTCTAACATTGGGAAAAATTATGTATCC   53 SGSSTNIGNNYVS  236TCTGGAAGCAGCACCAACATTGGGAATAATTATGTATCC   54 SGSISNIGDNYVS  237TCTGGAAGCATCTCCAACATTGGGGATAATTATGTATCC   55 SGSSSNIGSKDVS  238TCTGGAAGCAGCTCCAACATTGGGAGTAAGGATGTATCA   56 SGSSSNIENNDVS  239TCTGGAAGCAGCTCCAACATTGAGAATAATGATGTATCG   57 SGSSSNIGNHYVS  240TCTGGAAGCAGCTCCAACATTGGGAATCATTATGTATCC   58 SGSSSNIGKDFVS  241TCTGGAAGCAGCTCCAACATTGGGAAGGATTTTGTCTCC   59 SGSTSNIGSNFVS  242TCTGGCAGTACTTCCAACATCGGAAGTAATTTTGTTTCC   60 SGSTSNIGHNYVS  243TCTGGAAGCACCTCCAACATTGGGCATAATTATGTATCC   61 SASSSNIGNNYVS  244TCTGCAAGCAGCTCCAACATTGGGAATAATTATGTATCC   62 SGSSSSIGNNYVS  245TCTGGAAGCAGCTCCAGCATTGGCAATAATTATGTATCC   63 SGSSSTIGNNYVS  246TCTGGAAGCAGCTCCACCATTGGGAATAATTATGTATCC   64 SGSSSNIENNYVS  247TCTGGAAGCAGCTCCAACATTGAAAATAATTATGTATCC   65 SGSSSNIGNQYVS  248TCTGGAAGCAGCTCCAACATTGGGAATCAGTATGTATCC   66 SGSSSNIGNNYVF  249TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATTC   67 SGSSSNIGRNYVS  250TCTGGAAGCAGCTCCAACATTGGGAGGAATTATGTCTCC   68 SGGSSNIGNYYVS  251TCTGGAGGCAGCTCCAACATTGGAAATTATTATGTATCG   69 SGSSSNIGDNYVS  252TCTGGAAGCAGCTCCAACATTGGAGATAATTATGTCTCC   70 SGGSSNIGINYVS  253TCTGGAGGCAGCTCCAACATTGGAATTAATTATGTATCC   71 SGGSSNIGKNYVS  254TCTGGAGGCAGCTCCAACATTGGGAAGAATTATGTATCC   72 SGSSSNIGKRSVS  255TCTGGAAGCAGCTCCAACATTGGGAAGAGATCTGTATCG   73 SGSRSNIGNNYVS  256TCTGGAAGCAGATCCAACATTGGGAATAACTATGTATCC   74 SGSSSNIGNNLVS  257TCGGGAAGCAGCTCCAACATTGGGAATAATCTTGTTTCC   75 SGSSSNIGINYVS  258TCTGGAAGCAGCTCCAACATTGGGATCAATTATGTATCC   76 SGSSSNIGNNFVS  259TCTGGAAGCAGCTCCAACATCGGGAATAATTTTGTATCC   77 SGTSSNIGRNFVS  260TCTGGAACCAGCTCCAACATTGGCAGAAATTTTGTATCC   78 SGRRSNIGNNYVS  261TCTGGAAGGAGGTCCAACATTGGAAATAATTATGTGTCC   79 SGGSFNIGNNYVS  262TCTGGAGGCAGCTTCAATATTGGGAATAATTATGTATCC   80 SGSTSNIGENYVS  263TCTGGAAGCACTTCCAACATTGGGGAGAATTATGTGTCC   81 SGSSSNIGSDYVS  264TCTGGAAGCAGCTCCAATATTGGGAGTGATTATGTATCC   82 SGTSSNIGSNYVS  265TCTGGAACCAGCTCCAACATTGGGAGTAATTATGTATCC   83 SGSSSNIGTNFVS  266TCTGGAAGCAGCTCCAACATTGGGACTAATTTTGTATCC   84 SGSSSNFGNNYVS  267TCTGGAAGCAGCTCCAACTTTGGGAATAATTATGTATCC   85 SGSTSNIGNNHVS  268TCTGGAAGCACCTCCAACATTGGGAATAATCATGTATCC   86 SGSSSNIGNDFVS  269TCTGGAAGCAGCTCCAACATTGGGAATGATTTTGTATCC   87 SGSSSDIGDNYVS  270TCTGGAAGCAGCTCCGACATTGGCGATAATTATGTGTCC   88 SGSSSNIGKYYVS  271TCTGGAAGCAGCTCCAACATTGGGAAATATTATGTATCC   89 SGSSSNIGGNYVS  272TCTGGAAGCAGCTCCAACATTGGCGGTAATTATGTATCC   90 SGSSSNTGNNYVS  273TCTGGAAGCAGCTCCAACACTGGGAATAATTATGTATCC   91 SGSSSNVGNNYVS  274TCTGGAAGCAGCTCCAACGTTGGGAATAATTATGTGTCT   92 SGSSSNIANNFVS  275TCTGGAAGCAGCTCCAACATTGCGAATAATTTTGTATCC   93 SGSSSNIGNDYVS  276TCTGGAAGCAGCTCCAACATTGGGAATGATTATGTATCC   94 SGSTSNIENNYVS  277TCTGGAAGCACCTCCAATATTGAGAATAATTATGTTTCC   95 SGGSSNIGNNDVS  278TCTGGAGGCAGCTCCAATATTGGCAATAATGATGTGTCC   96 SGSTSNIGNHYVS  279TCTGGAAGCACCTCCAACATTGGGAATCATTATGTATCC   97 SGSSSNIGDNDVS  280TCAGGAAGCAGCTCCAATATTGGGGATAATGATGTATCC   98 SGYSSNIGNNYVS  281TCTGGATACAGCTCCAACATTGGGAATAATTATGTATCC   99 SGSGSNIGNNFVS  282TCTGGAAGCGGCTCCAACATTGGAAATAATTTTGTATCC  100 SGSSSNIWNNYVS  283TCTGGAAGCAGCTCCAACATTTGGAATAATTATGTATCC  101 FGSSSNIGNNYVS  284TTTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  102 SGSSSNIEKNYVS  285TCTGGAAGCAGCTCCAACATTGAGAAGAATTATGTATCC  103 SGSRSNIGNYYVS  286TCTGGAAGTAGATCCAATATTGGAAATTATTATGTATCC  104 SGTKSNIGNNYVS  287TCTGGAACCAAGTCAAACATTGGGAATAATTATGTATCT  105 SGSTSNIGNYYVS  288TCTGGAAGCACCTCCAACATTGGGAATTATTATGTATCC  106 SGTSSNIGNNYVA  289TCTGGAACCAGCTCCAACATTGGGAATAATTATGTGGCC  107 PGTSSNIGNNYVS  290CCTGGAACCAGCTCCAACATTGGGAATAATTATGTATCC  108 SGSTSNIGINYVS  291TCCGGAAGCACCTCCAACATTGGGATTAATTATGTATCC  109 SGSSSNIGSNLVS  292TCTGGAAGCAGCTCCAACATTGGGAGTAATCTGGTATCC  110 SGSSSNIENNHVS  293TCTGGAAGCAGCTCCAACATTGAGAATAATCATGTATCC  111 SGTRSNIGNNYVS  294TCTGGAACCAGGTCCAACATCGGCAATAATTATGTTTCG  112 SGSTSNIGDNYVS  295TCTGGAAGCACCTCCAACATTGGGGACAATTATGTTTCC  113 SGGSSNIGKNFVS  296TCTGGAGGCAGTTCCAACATTGGGAAGAATTTTGTATCC  114 SGSRSDIGNNYVS  297TCTGGAAGCAGGTCCGACATTGGGAATAATTATGTATCC  115 SGTSSNIGNNDVS  298TCTGGAACTAGCTCCAACATTGGGAATAATGATGTATCC  116 SGSSSNIGSKYVS  299TCTGGAAGCAGCTCCAACATTGGGAGTAAATATGTATCA  117 SGSSFNIGNNYVS  300TCTGGAAGCAGCTTCAACATTGGGAATAATTATGTATCC  118 SGSSSNIGNTYVS  301TCTGGAAGCAGCTCCAACATTGGGAATACTTATGTATCC  119 SGSSSNIGDNHVS  302TCTGGAAGCAGCTCCAATATTGGGGATAATCATGTATCC  120 SGSSSNIGNNHVS  303TCTGGAAGCAGCTCCAACATTGGCAATAATCATGTTTCC  121 SGSTSNIGNNDVS  304TCTGGAAGCACCTCCAACATTGGGAATAATGATGTATCC  122 SGSRSNVGNNYVS  305TCTGGAAGCAGATCCAACGTTGGCAATAATTATGTTTCA  123 SGGTSNIGKNYVS  306TCCGGAGGCACCTCCAACATTGGGAAGAATTATGTGTCT  124 SGSSSNIADNYVS  307TCTGGAAGCAGCTCCAACATTGCCGATAATTATGTTTCC  125 SGSSSNIGANYVS  308TCTGGAAGCAGCTCCAACATTGGCGCCAATTATGTATCC  126 SGSSSNIGSNYVA  309TCTGGAAGCAGCTCCAACATTGGGAGTAATTATGTGGCC  127 SGSSSNIGNNFLS  310TCTGGAAGCAGCTCCAACATTGGGAACAATTTTCTCTCC  128 SGRSSNIGKNYVS  311TCTGGAAGAAGCTCCAACATTGGGAAGAATTATGTATCC  129 SGSSPNIGANYVS  312TCTGGAAGCAGCCCCAACATTGGGGCTAATTATGTATCC  130 SGSSSNIGPNYVS  313TCCGGAAGCAGCTCCAACATTGGGCCTAATTATGTGTCC  131 SGSSSTIGNNYIS  314TCTGGAAGCAGCTCCACCATTGGGAATAATTATATATCC  132 SGSSSNIGNYFVS  315TCTGGAAGCAGCTCCAACATTGGGAATTATTTTGTATCC  133 SGSRSNIGNNFVS  316TCTGGAAGCCGCTCCAACATTGGTAATAATTTTGTATCC  134 SGGSSNIGSNFVS  317TCTGGAGGCAGCTCCAACATTGGGAGTAATTTTGTATCC  135 SGSSSNIGYNYVS  318TCTGGAAGCAGCTCCAACATTGGGTATAATTATGTATCC  136 SGTSSNIENNYVS  319TCTGGAACCAGCTCGAACATTGAGAACAATTATGTATCC  137 SGSSSNIGNYYVS  320TCTGGAAGTAGCTCCAACATTGGGAATTATTATGTATCC  138 SGSTSNIGKNYVS  321TCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATCC  139 SGSSSNIGTYYVS  322TCTGGAAGCAGTTCCAACATTGGGACTTATTATGTCTCT  140 SGSSSNVGKNYVS  323TCTGGAAGCAGCTCCAACGTTGGGAAAAATTATGTATCT  141 SGSTSNIGDNFVS  324TCTGGAAGCACCTCCAACATTGGGGATAATTTTGTATCC  142 SGSTSNIGTNYVS  325TCTGGAAGCACCTCCAACATTGGAACTAATTATGTTTCC  143 SGGTSNIGNNYVS  326TCTGGAGGTACTTCCAACATTGGGAATAATTATGTCTCC  144 SGSYSNIGNNYVS  327TCTGGAAGCTACTCCAATATTGGGAATAATTATGTATCC  145 SGSSSNIEDNYVS  328TCTGGAAGCAGCTCCAACATTGAAGATAATTATGTATCC  146 SGSSSNIGKHYVS  329TCTGGAAGCAGCTCCAACATTGGGAAACATTATGTATCC  147 SGSGSNIGSNYVS  330TCCGGTTCCGGCTCAAACATTGGAAGTAATTATGTCTCC  148 SGSSSNIGNNYIS  331TCTGGAAGCAGCTCCAACATTGGAAATAATTATATATCA  149 SGASSNIGNNYVS  332TCTGGAGCCAGTTCCAACATTGGGAATAATTATGTTTCC  150 SGRTSNIGNNYVS  333TCTGGACGCACCTCCAACATCGGGAACAATTATGTATCC  151 SGGSSNIGSNYVS  334TCTGGAGGCAGCTCCAATATTGGGAGTAATTACGTATCC  152 SGSGSNIGNNYVS  335TCTGGAAGCGGCTCCAACATTGGGAATAATTATGTATCC  153 SGSTSNIGSNYVS  336TCTGGAAGCACCTCCAACATTGGGAGTAATTATGTATCC  154 SGSSSSIGNNYVA  337TCTGGAAGCAGCTCCAGCATTGGGAATAATTATGTGGCG  155 SGSSSNLGNNYVS  338TCTGGAAGCAGTTCCAACCTTGGAAATAATTATGTATCC  156 SGTSSNIGKNYVS  339TCTGGAACCAGCTCCAACATTGGGAAAAATTATGTATCC  157 SGSSSDIGNKYIS  340TCTGGAAGCAGCTCCGATATTGGGAACAAGTATATATCC  158 SGSSSNIGSNYIS  341TCTGGAAGCAGCTCCAACATTGGAAGTAATTACATATCC  159 SGSTSNIGANYVS  342TCTGGAAGCACCTCCAACATTGGGGCTAACTATGTGTCC  160 SGSSSNIGNKYVS  343TCTGGAAGCAGCTCCAACATTGGGAATAAGTATGTATCC  161 SGSSSNIGNNYGS  344TCTGGAAGCAGCTCCAACATTGGGAATAATTATGGATCC  162 SGSTSNIANNYVS  345TCTGGAAGCACCTCCAACATTGCGAATAATTATGTATCC  163 SGSYSNIGSNYVS  346TCTGGAAGCTACTCCAATATTGGGAGTAATTATGTATCC  164 SGSSSNIGSNFVS  347TCTGGAAGCAGCTCCAACATTGGGAGTAATTTTGTATCC  165 SGSSSNLENNYVS  348TCTGGAAGCAGCTCCAATCTTGAGAATAATTATGTATCC  166 SGSISNIGSNYVS  349TCTGGAAGCATCTCCAATATTGGCAGTAATTATGTATCC  167 SGSSSDIGSNYVS  350TCTGGAAGCAGCTCCGACATTGGGAGTAATTATGTATCC  168 SGSSSNIGTNYVS  351TCTGGAAGCAGCTCCAACATTGGGACTAATTATGTATCC  169 SGSSSNIGKNFVS  352TCTGGAAGCAGCTCCAACATTGGGAAGAATTTTGTATCC  170 SGSSSNIGNNFIS  353TCTGGAAGCAGCTCCAACATTGGGAATAATTTTATATCC  171 SGGSSNIGNNYVS  354TCTGGAGGCAGCTCCAACATTGGCAATAATTATGTTTCC  172 SGSSSNIGENYVS  355TCTGGAAGCAGCTCCAACATTGGGGAGAATTATGTATCC  173 SGSSSNIGNNFVA  356TCTGGAAGCAGCTCCAATATTGGGAATAATTTTGTGGCC  174 SGGSSNIGNNYVA  357TCTGGAGGCAGCTCCAACATTGGGAATAATTATGTAGCC  175 SGSSSHIGNNYVS  358TCTGGAAGCAGCTCCCACATTGGAAATAATTATGTATCC  176 SGSSSNIGSNDVS  359TCTGGAAGCAGCTCCAATATTGGAAGTAATGATGTATCG  177 SGSSSNIGNNYVT  360TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTAACC  178 SGSSSNIGNNPVS  361TCTGGAAGCAGCTCCAACATTGGGAATAATCCTGTATCC  179 SGGSSNIGNHYVS  362TCTGGAGGCAGCTCCAATATTGGGAATCATTATGTATCC  180 SGTSSNIGNNYVS  363TCTGGAACCAGCTCCAACATTGGGAATAATTATGTATCC  181 SGSSSNIGSNYVS  364TCTGGAAGCAGCTCCAACATTGGAAGTAATTATGTCTCG  182 SGGTSNIGSNYVS  365TCTGGAGGCACCTCCAACATTGGAAGTAATTATGTATCC  183 SGSKSNIGNNYVS  366TCTGGAAGCAAGTCCAACATTGGGAATAATTATGTATCC  184 SGRSSNIGNNYVS  367TCTGGAAGAAGCTCCAACATTGGGAATAATTATGTATCG  185 SGSSSNVGSNYVS  368TCTGGAAGCAGCTCCAACGTTGGGAGTAATTATGTTTCC  186 SGSTSNIGNNFVS  369TCTGGAAGCACCTCCAATATTGGGAATAATTTTGTATCC  187 SGSNFNIGNNYVS  370TCTGGAAGCAACTTCAACATTGGGAATAATTATGTCTCC  188 SGSTSNIGYNYVS  371TCTGGAAGCACCTCCAATATTGGATATAATTATGTATCC  189 SGSSSNIVSNYVS  372TCTGGAAGCAGCTCCAATATTGTAAGTAATTATGTATCC  190 SGTSSNIGNNFVS  373TCTGGAACCAGCTCCAACATTGGGAATAATTTTGTATCC  191 SGSSSNIGRNFVS  374TCTGGAAGCAGCTCCAACATTGGGAGGAATTTTGTGTCC  192 SGTTSNIGNNYVS  375TCTGGAACGACCTCCAACATTGGGAATAATTATGTCTCC  193 SGSSSNIGNNDVS  376TCTGGAAGCAGCTCCAACATTGGGAATAATGATGTATCC  194 SGSSSNIGNHDVS  377TCTGGAAGCAGCTCCAACATTGGGAATCATGATGTATCC  195 SGSSSNIGSSHVS  378TCTGGAAGCAGCTCCAACATTGGAAGTAGTCATGTATCC  196 SGSSSNIGIHYVS  379TCTGGAAGCAGCTCCAACATTGGGATTCATTATGTATCC  197 SGGGSNIGYNYVS  380TCTGGAGGCGGCTCCAACATTGGCTATAATTATGTCTCC  198 SGSSSNIGDHYVS  381TCTGGAAGCAGCTCCAACATTGGGGATCATTATGTGTCG  199 SGSSSNLGKNYVS  382TCTGGAAGCAGCTCCAACCTTGGGAAGAATTATGTATCT  200 SGSSSNIGDNFVS  383TCTGGAAGCAGCTCCAACATTGGCGATAATTTTGTATCC  201 SGSTSNIEKNYVS  384TCTGGAAGCACCTCCAACATTGAGAAAAACTATGTATCG  202 SGSSSNIGKDYVS  385TCTGGAAGCAGCTCCAACATTGGGAAGGATTATGTATCC  203 SGSSSNIGKNYVS  386TCTGGAAGCAGCTCCAACATTGGGAAGAATTATGTATCC  204 SGSSSNIGNNYVS  387TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  205 SGSSSNIGNNYAS  388TCTGGAAGCAGCTCCAACATTGGGAATAATTATGCCTCC  206 SGISSNIGNNYVS  389TCTGGAATCAGCTCCAACATTGGGAATAATTATGTATCC  207 TGSSSNIGNNYVS  390ACTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  208 SGTSSNIGNNHVS  391TCTGGAACCAGCTCCAACATTGGGAATAATCATGTTTCC  209 SGSRSNIGKNYVS  392TCTGGAAGTCGTTCCAACATTGGGAAAAATTATGTATCC IGLV1-51-L2  393 DNNKRPP  544GACAATAATAAGCGACCCCCA  394 ENNRRPS  545 GAGAATAATAGGCGACCCTCA  395DNNKQPS  546 GACAATAATAAGCAACCCTCA  396 DNNKRPL  547GACAATAACAAGCGACCCTTG  397 DNDKRPA  548 GACAATGATAAGCGACCCGCA  398DNHERPS  549 GACAATCATGAGCGACCCTCA  399 ENRKRPS  550GAAAACCGTAAGCGACCCTCA  400 DNDQRPS  551 GACAATGATCAGCGACCCTCA  401ENYKRPS  552 GAGAATTATAAGCGACCCTCA  402 ENTKRPS  553GAAAATACTAAGCGACCCTCA  403 DTEKRPS  554 GACACTGAGAAGAGGCCCTCA  404DNDKRPP  555 GACAATGATAAGCGACCCCCA  405 DHNKRPS  556GACCATAATAAGCGACCCTCA  406 GNNERPS  557 GGCAATAATGAGCGACCCTCA  407DTSKRPS  558 GACACTAGTAAGCGACCCTCA  408 EYNKRPS  559GAATATAATAAGCGCCCCTCA  409 ENIKRPS  560 GAAAATATTAAGCGACCCTCA  410DNVKRPS  561 GACAATGTTAAGCGACCCTCA  411 ENDKRSS  562GAAAACGATAAACGATCCTCA  412 ENNKRHS  563 GAAAATAATAAGCGACACTCA  413GNDQRPS  564 GGAAATGATCAGCGACCCTCA  414 DNDRRPS  565GACAATGATAGGCGACCCTCA  415 DNHKRPS  566 GACAATCATAAGCGGCCCTCA  416DNNDRPS  567 GACAATAATGACCGACCCTCA  417 ENNQRPS  568GAGAATAATCAGCGACCCTCA  418 DNNQRPS  569 GACAATAATCAGCGACCCTCA  419ENVKRPS  570 GAGAATGTTAAGCGACCCTCA  420 DTYKRPS  571GACACTTATAAGAGACCCTCA  421 NNNNRPS  572 AACAATAATAACCGACCCTCA  422GNNNRPS  573 GGCAATAATAATCGACCCTCA  423 ENDQRPS  574GAAAATGATCAGCGACCCTCA  424 DNNKRAS  575 GACAATAATAAGCGAGCCTCA  425DNDKRPL  576 GACAATGATAAGCGACCCTTA  426 DTDERPS  577GACACTGATGAGCGACCTTCA  427 DNRKRPS  578 GACAATAGGAAGCGACCCTCA  428DNDARPS  579 GACAATGATGCTCGACCCTCA  429 DNNKRLS  580GACAATAATAAGCGACTCTCA  430 DNDKRAS  581 GACAATGATAAGCGAGCCTCA  431DNTERPS  582 GACAATACTGAGCGACCCTCA  432 DNNIRPS  583GACAATAATATTCGACCCTCA  433 DNKRRPS  584 GACAATAAGAGGCGACCCTCA  434DDNNRPS  585 GACGATAATAACCGACCCTCA  435 ANNRRPS  586GCGAATAATCGACGACCCTCA  436 DNDKRLS  587 GACAATGATAAGCGACTGTCA  437DNNKRPA  588 GACAATAATAAGCGACCCGCA  438 DNYRRPS  589GACAATTATAGACGTCCCTCA  439 ANDQRPS  590 GCCAATGATCAGCGACCCTCA  440DNDKRRS  591 GACAATGATAAGCGACGCTCA  441 DKNERPS  592GACAAGAATGAGCGACCCTCA  442 DNKERPS  593 GACAATAAGGAGCGACCCTCA  443DNNKGPS  594 GACAATAATAAGGGACCCTCA  444 ENDRRPS  595GAAAATGATAGACGACCCTCA  445 ENDERPS  596 GAAAATGATGAGCGACCCTCA  446QNNKRPS  597 CAAAATAATAAGCGACCCTCA  447 DNRERPS  598GACAATCGTGAGCGACCCTCA  448 DNNRRPS  599 GACAATAATAGACGACCCTCA  449GNNRRPS  600 GGAAATAATAGGCGACCCTCA  450 DNDNRPS  601GACAATGATAACCGACCCTCA  451 EDNKRPS  602 GAAGATAATAAGCGACCCTCA  452DDDERPS  603 GACGATGATGAGCGGCCCTCA  453 ASNKRPS  604GCAAGTAATAAGCGACCCTCA  454 DNNKRSS  605 GACAATAATAAGCGATCCTCA  455QNNERPS  606 CAAAATAATGAGCGACCCTCA  456 DDDRRPS  607GACGATGATAGGCGACCCTCA  457 NNDKRPS  608 AACAATGATAAGCGACCCTCA  458DNNNRPS  609 GACAATAATAACCGACCCTCA  459 DNNVRPS  610GACAATAATGTGCGACCCTCA  460 ENNERPS  611 GAAAATAATGAGCGACCCTCA  461DNNHRPS  612 GACAATAATCACCGACCCTCA  462 DNDERPS  613GACAATGATGAGCGCCCCTCG  463 DNIRRPS  614 GACAATATCCGGCGACCCTCA  464DFNKRPS  615 GACTTTAATAAGCGACCCTCA  465 ETNKRPS  616GAAACTAATAAGCGACCCTCA  466 NDNKRPS  617 AACGATAATAAGCGACCCTCA  467DDNKRPS  618 GACGATAATAAGCGACCCTCA  468 DNYKRPS  619GACAATTATAAGCGACCCTCA  469 HNNKRPS  620 CACAATAATAAGCGACCCTCA  470DNHQRPS  621 GACAATCATCAGCGACCCTCA  471 DNYKRAS  622GACAATTATAAGCGAGCCTCA  472 DNIKRPS  623 GACAATATTAAGCGACCCTCA  473DTHKRPS  624 GACACTCATAAGCGACCCTCA  474 DTNRRPS  625GACACTAATAGGCGACCCTCT  475 DTNQRPS  626 GACACTAATCAGCGACCCTCA  476ESDKRPS  627 GAAAGTGATAAGCGACCCTCA  477 DNDKRSS  628GACAATGATAAGCGATCTTCG  478 GSNKRPS  629 GGCAGTAATAAGCGACCCTCA  479DNNKRVS  630 GACAATAACAAGCGAGTTTCA  480 NNNRRPS  631AACAATAATAGGCGACCCTCA  481 DNFKRPS  632 GACAATTTTAAGCGACCCTCA  482ENDKRPS  633 GAAAATGATAAACGACCCTCA  483 ENNKRLS  634GAAAATAATAAGCGACTCTCA  484 ADNKRPS  635 GCAGATAATAAGCGACCCTCA  485EDNERPS  636 GAAGATAATGAGCGCCCCTCA  486 DTDQRPS  637GACACTGATCAGCGACCCTCA  487 DNYQRPS  638 GACAATTATCAGCGACCCTCA  488DENKRPS  639 GACGAGAATAAGCGACCCTCA  489 DTNKRPS  640GACACTAATAAGCGACCCTCA  490 DDYRRPS  641 GACGATTATCGGCGACCCTCA  491DNDKRHS  642 GACAACGATAAGCGGCACTCA  492 ENDNRPS  643GAAAATGATAATCGACCCTCA  493 DDNERPS  644 GACGATAATGAGCGCCCCTCA  494DNKKRPS  645 GACAATAAGAAGCGACCCTCA  495 DVDKRPS  646GACGTTGATAAGCGACCCTCA  496 ENKKRPS  647 GAAAATAAAAAACGACCCTCT  497VNDKRPS  648 GTCAATGATAAGCGACCCTCA  498 DNDHRPS  649GACAATGATCACCGACCCTCA  499 DINKRPS  650 GACATTAATAAGCGACCCTCA  500ANNERPS  651 GCCAATAATGAGCGACCCTCA  501 DNENRPS  652GACAATGAAAACCGACCGTCA  502 GDDKRPS  653 GGCGATGATAAGCGACCCTCA  503ANNQRPS  654 GCCAATAATCAGCGACCTTCA  504 DDDKRPS  655GACGATGATAAGCGACCCTCA  505 YNNKRPS  656 TACAATAATAAGCGGCCCTCA  506EDDKRPS  657 GAAGATGATAAGCGACCCTCA  507 ENNNRPS  658GAAAACAATAACCGACCCTCG  508 DNNLRPS  659 GACAATAATCTGCGACCCTCA  509ESNKRPS  660 GAGAGTAACAAGCGACCCTCA  510 DTDKRPS  661GACACTGATAAGCGGCCCTCA  511 DDDQRPS  662 GACGATGATCAGCGACCCTCA  512VNNKRPS  663 GTGAATAATAAGAGACCCTCC  513 DDYKRPS  664GACGATTATAAGCGACCCTCA  514 DNTKRPS  665 GACAATACTAAGCGACCCTCA  515DDTERPS  666 GACGATACTGAGCGACCCTCA  516 GNDKRPS  667GGCAATGATAAGCGACCCTCA  517 DNEKRPS  668 GACAATGAAAAGCGACCCTCA  518DNDDRPS  669 GACAATGATGACCGACCCTCA  519 DDNRRPS  670GACGATAATAGGCGTCCCTCA  520 GNNKRPS  671 GGCAATAATAAGCGACCCTCA  521ANDKRPS  672 GCCAATGATAAGCGACCCTCA  522 DNNKRHS  673GACAATAATAAGCGACACTCA  523 DDNQRPS  674 GACGACAATCAGCGACCCTCA  524GNDRRPS  675 GGCAATGATAGGCGACCCTCA  525 DNHNRPS  676GACAATCATAACCGACCCTCA  526 DNYERPS  677 GACAATTATGAGCGACCCTCA  527ENNKRSS  678 GAAAATAATAAGCGATCCTCA  528 DDHKRPS  679GACGATCATAAGCGGCCCTCA  529 DNNKRRS  680 GACAATAATAAACGACGTTCA  530DNDKRPS  681 GACAATGATAAGCGACCGTCA  531 DKNKRPS  682GACAAGAATAAGCGACCCTCA  532 DNNKRPS  683 GACAATAATAAGCGACCCTCA  533DIDKRPS  684 GACATTGATAAGCGACCCTCA  534 DDKKRPS  685GACGATAAGAAGCGACCCTCA  535 ANNKRPS  686 GCCAATAATAAGCGACCCTCA  536DNDKGPS  687 GACAATGATAAGGGACCCTCA  537 EDNRRPS  688GAAGATAATAGGCGACCCTCA  538 ENNKRPS  689 GAGAATAATAAGCGACCCTCA  539NNNKRPS  690 AACAATAATAAGCGACCCTCA  540 DNNERPS  691GACAATAATGAGCGACCCTCA  541 DNIQRPS  692 GACAATATTCAGCGACCCTCA  542DNNYRPS  693 GACAATAATTACCGACCCTCA  543 DNYNRPS  694GACAATTATAACCGACCCTCA IGLV1-51-L3  695 CGTWDTSLSAVVF 1431TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTGTTC  696 CGTWDTSLSAGVF 1432TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGTGTTC  697 CGTWDTSLSAWVF 1433TGCGGAACATGGGATACCAGCCTGAGTGCTTGGGTGTTC  698 CGTWDRSLSAGVF 1434TGCGGAACATGGGATAGGAGCCTGAGTGCGGGGGTGTTC  699 CGTWDRSLSAWVF 1435TGCGGAACATGGGATAGGAGCCTGAGTGCTTGGGTATTT  700 CGTWDTSLSGGVF 1436TGCGGAACATGGGATACCAGCCTGAGTGGTGGGGTGTTC  701 CGTWDTSLRAGVF 1437TGCGGAACATGGGATACTAGCCTGCGTGCTGGCGTCTTC  702 CGTWDRSLSVWVF 1438TGCGGAACATGGGATAGGAGCCTGAGTGTTTGGGTGTTC  703 CGTWDTSLSVVVF 1439TGCGGAACATGGGATACCAGTCTGAGTGTTGTGGTCTTC  704 CGTWDTSLSAAVF 1440TGCGGAACGTGGGATACCAGCCTGAGTGCTGCGGTGTTC  705 CGAWDTSLSAGVF 1441TGCGGAGCATGGGATACCAGCCTGAGTGCTGGAGTGTTC  706 CATWDTSLSAVVF 1442TGCGCAACATGGGATACCAGCCTGAGTGCTGTGGTATTC  707 CATWDTSLSAGVF 1443TGCGCAACATGGGATACCAGCCTGAGTGCTGGTGTGTTC  708 CGTWESSLSAWVF 1444TGTGGAACATGGGAGAGCAGCCTGAGTGCTTGGGTGTTC  709 CGTWDTTLSAGVF 1445TGCGGAACATGGGATACCACCCTGAGTGCGGGTGTCTTC  710 CGTWDTSLSVWVF 1446TGCGGAACATGGGATACTAGCCTGAGTGTGTGGGTGTTC  711 CGTWDTSLSVGVF 1447TGCGGAACATGGGATACTAGCCTGAGTGTTGGGGTGTTC  712 CGTWDTSLSTGVF 1448TGCGGAACATGGGACACCAGTCTGAGCACTGGCGTCTTC  713 CGTWDTSLSGVVF 1449TGCGGAACATGGGATACCAGCCTGAGTGGTGTGGTCTTC  714 CGTWDTSLSAYVF 1450TGCGGAACATGGGATACCAGCCTGAGTGCTTATGTCTTC  715 CGTWDTSLSAEVF 1451TGCGGAACATGGGATACCAGCCTGAGTGCTGAGGTGTTC  716 CGTWDTGLSAGVF 1452TGCGGAACATGGGATACCGGCCTGAGTGCTGGGGTATTC  717 CGTWDRSLSAYVF 1453TGCGGAACGTGGGATAGGAGCCTGAGTGCTTATGTCTTC  718 CGTWDRSLSAVVF 1454TGCGGAACATGGGATAGGAGCCTCAGTGCCGTGGTATTC  719 CGTWDNTLSAWVF 1455TGCGGAACATGGGATAACACCCTGAGTGCGTGGGTGTTC  720 CGTWDNRLSAGVF 1456TGCGGAACATGGGATAACAGGCTGAGTGCTGGGGTGTTC  721 CGTWDISLSAWVF 1457TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGTGTTC  722 CGTWHSSLSAGVF 1458TGCGGAACATGGCATAGCAGCCTGAGTGCTGGGGTATTC  723 CGTWGSSLSAWVF 1459TGCGGAACATGGGGTAGCAGTTTGAGTGCTTGGGTGTTC  724 CGTWESSLSGWVF 1460TGCGGAACATGGGAGAGCAGCCTGAGTGGTTGGGTGTTC  725 CGTWESSLSAVVF 1461TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTGGTTTTC  726 CGTWDYSLSAVVF 1462TGCGGAACATGGGATTACAGCCTGAGTGCTGTGGTATTC  727 CGTWDYSLSAGVF 1463TGCGGAACATGGGATTACAGCCTGAGTGCTGGGGTATTC  728 CGTWDVSLSVGVF 1464TGCGGAACATGGGATGTCAGCCTGAGTGTTGGAGTGTTC  729 CGTWDTTLSAVVF 1465TGCGGAACATGGGATACCACCCTGAGTGCTGTGGTTTTC  730 CGTWDTTLNIGVF 1466TGCGGAACATGGGATACCACTCTGAATATTGGGGTGTTC  731 CGTWDTSLTAVVF 1467TGCGGAACATGGGATACCAGCCTGACTGCTGTGGTATTC  732 CGTWDTSLTAAVF 1468TGCGGAACCTGGGATACCAGCCTGACTGCTGCTGTGTTC  733 CGTWDTSLSVGLF 1469TGCGGCACATGGGATACCAGCCTGAGTGTGGGGCTATTC  734 CGTWDTSLSGRVF 1470TGCGGAACCTGGGATACCAGCCTGAGTGGTAGGGTGTTC  735 CGTWDTSLSGAVF 1471TGCGGAACATGGGATACCAGCCTGAGTGGTGCAGTGTTC  736 CGTWDTSLSAGLF 1472TGCGGAACATGGGATACCAGCCTGAGTGCTGGCCTGTTC  737 CGTWDTSLSAGGVF 1473TGCGGAACATGGGATACCAGCCTGAGTGCTGGAGGGGTCTTC  738 CGTWDTSLRAYVF 1474TGCGGAACATGGGATACCAGCCTGCGTGCTTATGTCTTC  739 CGTWDTSLRAWVF 1475TGCGGAACATGGGATACTAGTTTGCGTGCTTGGGTATTC  740 CGTWDTSLNTGVF 1476TGCGGAACATGGGATACCAGCCTGAATACTGGGGTATTC  741 CGTWDTSLNIWVF 1477TGCGGAACATGGGATACCAGCCTGAATATTTGGGTGTTC  742 CGTWDTSLNIGVF 1478TGCGGAACATGGGATACAAGCCTGAATATTGGGGTGTTC  743 CGTWDTSLIAVVF 1479TGCGGAACATGGGATACCAGCCTGATTGCTGTGGTGTTC  744 CGTWDRSLSGWVF 1480TGCGGAACGTGGGATAGGAGCCTGAGTGGTTGGGTGTTC  745 CGTWDNRLSGWVF 1481TGCGGAACATGGGATAACAGGCTGAGTGGTTGGGTGTTC  746 CGTWDKSLSAVVF 1482TGCGGAACGTGGGATAAGAGCCTGAGTGCTGTGGTCTTC  747 CGTWDKGLSAWVF 1483TGCGGAACATGGGATAAAGGCCTGAGTGCTTGGGTGTTC  748 CGTWDISLSAGVF 1484TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTGTTC  749 CGTWDESLSGGEVVF 1485TGCGGAACATGGGATGAGAGCCTGAGTGGTGGCGAGGTGGTCTTC  750 CGTWDASLSAWVF 1486TGCGGAACATGGGATGCCAGCCTGAGTGCCTGGGTGTTC  751 CGTWDAGLSAWVF 1487TGCGGAACTTGGGATGCCGGCCTGAGTGCTTGGGTGTTC  752 CGAWDTSLSAWVF 1488TGCGGAGCATGGGATACCAGCCTGAGTGCTTGGGTGTTC  753 CGAWDTSLSAVVF 1489TGCGGAGCATGGGATACCAGCCTGAGTGCTGTGGTGTTC  754 CGAWDTSLRAGVF 1490TGCGGAGCATGGGATACCAGCCTGCGTGCTGGGGTTTTC  755 CATWDTSVSAWVF 1491TGCGCAACATGGGATACCAGCGTGAGTGCTTGGGTGTTC  756 CATWDTSLSAWVF 1492TGCGCAACATGGGATACCAGCCTGAGTGCGTGGGTGTTC  757 CATWDNTLSAGVF 1493TGCGCAACATGGGACAACACCCTGAGTGCTGGGGTGTTC  758 CAAWDRSLSVWVF 1494TGCGCAGCATGGGATAGGAGCCTGAGTGTTTGGGTGTTC  759 CYTWHSSLRGGVF 1495TGCTACACATGGCATTCCAGTCTGCGTGGTGGGGTGTTC  760 CVTWTSSPSAWVF 1496TGCGTAACGTGGACTAGTAGCCCGAGTGCTTGGGTGTTC  761 CVTWRGGLVLF 1497TGCGTGACATGGCGTGGTGGCCTTGTGTTGTTC  762 CVTWDTSLTSVVL 1498TGCGTAACATGGGATACCAGCCTGACTTCTGTGGTACTC  763 CVTWDTSLSVYWVF 1499TGCGTAACATGGGATACCAGCCTGAGTGTTTATTGGGTGTTC  764 CVTWDTSLSAWVF 1500TGCGTTACATGGGATACCAGCCTGAGTGCCTGGGTGTTC  765 CVTWDTDLSVALF 1501TGCGTCACATGGGATACCGACCTCAGCGTTGCGCTCTTC  766 CVTWDRSLSGWVF 1502TGCGTAACATGGGATAGGAGCCTGAGTGGTTGGGTGTTC  767 CVTWDRSLREVLF 1503TGCGTAACATGGGATCGCAGCCTGAGAGAGGTGTTATTC  768 CVTWDRSLRAVVF 1504TGCGTAACATGGGATCGCAGCCTGAGAGCGGTGGTATTC  769 CVTWDRSLDAGVF 1505TGCGTAACATGGGACAGGAGCCTCGATGCTGGGGTTTTC  770 CVTWDNTLSAGVF 1506TGCGTGACATGGGATAACACCCTGAGTGCTGGGGTCTTC  771 CVTWDNNLFGVVF 1507TGCGTAACATGGGATAACAACCTGTTTGGTGTGGTCTTC  772 CVSWDTSLSGAVF 1508TGCGTATCATGGGATACCAGCCTGAGTGGTGCGGTATTC  773 CVSWDTSLSAGVF 1509TGCGTCTCATGGGATACCAGCCTGAGTGCTGGGGTATTC  774 CTTWFRTPSDVVF 1510TGCACAACATGGTTTAGGACTCCGAGTGATGTGGTCTTC  775 CTTWFRTASDVVF 1511TGCACAACATGGTTTAGGACTGCGAGTGATGTGGTCTTC  776 CTTWDYGLSVVF 1512TGCACAACGTGGGATTACGGTCTGAGTGTCGTCTTC  777 CTARDTSLSPGGVF 1513TGCACAGCAAGGGATACCAGCCTGAGTCCTGGCGGGGTCTTC  778 CSTWNTRPSDVVF 1514TGCTCAACATGGAATACGAGGCCGAGTGATGTGGTGTTC  779 CSTWESSLTTVVF 1515TGTTCAACATGGGAGAGCAGTTTGACTACTGTGGTCTTC  780 CSTWDTSLTNVLF 1516TGCTCAACATGGGATACCAGCCTCACTAATGTGCTATTC  781 CSTWDTSLSGVVF 1517TGCTCAACATGGGATACCAGCCTGAGTGGAGTAGTCTTC  782 CSTWDHSLKAALF 1518TGCTCAACATGGGATCACAGCCTGAAAGCTGCACTGTTC  783 CSTWDARLSVRVF 1519TGCTCAACCTGGGATGCGAGGCTGAGTGTCCGGGTGTTC  784 CSSYTSSSTWVF 1520TGCTCCTCATATACAAGCAGCAGCACTTGGGTGTTC  785 CSSYATRGLRVLF 1521TGCAGCTCATACGCAACCCGCGGCCTTCGTGTGTTGTTC  786 CSSWDATLSVRIF 1522TGTTCATCATGGGACGCCACCCTGAGTGTTCGCATATTC  787 CQVWEGSSDHWVF 1523TGTCAGGTGTGGGAGGGTAGTAGTGATCATTGGGTGTTC  788 CQTWDNRLSAVVF 1524TGCCAAACCTGGGATAACAGACTGAGTGCTGTGGTGTTC  789 CQTWDHSLHVGVF 1525TGTCAAACGTGGGATCACAGCCTGCATGTTGGGGTGTTC  790 CQSYDDILNVWVL 1526TGCCAGTCCTATGACGACATCTTGAATGTTTGGGTCCTT  791 CNTWDKSLTSELF 1527TGCAATACATGGGATAAGAGTTTGACTTCTGAACTCTTC  792 CLTWDRSLNVRVF 1528TGCTTAACATGGGATCGCAGCCTGAATGTGAGGGTGTTC  793 CLTWDHSLTAYVF 1529TGCCTAACATGGGACCACAGCCTGACTGCTTATGTCTTC  794 CLTRDTSLSAPVF 1530TGCTTAACAAGGGATACCAGTCTGAGTGCCCCTGTGTTC  795 CKTWESGLNFGHVF 1531TGCAAAACATGGGAAAGTGGCCTTAATTTTGGCCACGTCTTC  796 CKTWDTSLSAVVF 1532TGCAAAACATGGGATACCAGCCTGAGTGCTGTGGTCTTC  797 CGVWDVSLGAGVF 1533TGCGGAGTCTGGGATGTCAGTCTGGGTGCTGGGGTGTTC  798 CGVWDTTPSAVLF 1534TGCGGAGTCTGGGATACCACCCCGAGTGCCGTTCTTTTC  799 CGVWDTTLSAVLF 1535TGCGGAGTCTGGGATACCACCCTGAGTGCCGTTCTTTTC  800 CGVWDTSLGVF 1536TGCGGAGTATGGGATACCAGCCTGGGGGTCTTC  801 CGVWDTNLGKWVF 1537TGCGGGGTATGGGATACCAACCTGGGTAAATGGGTTTTC  802 CGVWDTGLDAGWVF 1538TGTGGAGTTTGGGATACTGGCCTGGATGCTGGTTGGGTGTTC  803 CGVWDNVLEAYVF 1539TGCGGAGTGTGGGATAACGTCCTGGAGGCCTATGTCTTC  804 CGVWDISLSANWVF 1540TGCGGAGTCTGGGATATCAGCCTGAGTGCTAATTGGGTGTTC  805 CGVWDHSLGIWAF 1541TGCGGAGTATGGGATCACAGCCTGGGGATTTGGGCCTTC  806 CGVWDDILTAEVF 1542TGCGGAGTTTGGGATGATATTCTGACTGCTGAAGTGTTC  807 CGVRDTSLGVF 1543TGCGGAGTTCGGGATACCAGCCTGGGGGTCTTC  808 CGTYDTSLPAWVF 1544TGCGGAACATACGATACGAGCCTGCCTGCTTGGGTGTTT  809 CGTYDNLVFGYVF 1545TGCGGAACTTACGATAATCTTGTATTTGGTTATGTCTTC  810 CGTYDDRLREVF 1546TGCGGAACATACGATGATAGACTCAGAGAGGTGTTC  811 CGTWVTSLSAGVF 1547TGCGGAACGTGGGTTACCAGCCTGAGTGCTGGGGTGTTC  812 CGTWVSSLTTVVF 1548TGCGGAACATGGGTTAGCAGCCTGACTACTGTAGTATTC  813 CGTWVSSLNVWVF 1549TGCGGAACATGGGTTAGCAGCCTGAACGTCTGGGTGTTC  814 CGTWVGRFWVF 1550TGCGGAACATGGGTTGGCAGGTTTTGGGTATTC  815 CGTWSGGPSGHWLF 1551TGCGGAACATGGTCTGGCGGCCCGAGTGGCCATTGGTTGTTC  816 CGTWSGGLSGHWLF 1552TGCGGAACATGGTCTGGCGGCCTGAGTGGCCATTGGTTGTTC  817 CGTWQTGREAVLF 1553TGCGGAACGTGGCAGACCGGCCGGGAGGCTGTCCTATTT  818 CGTWQSRLRWVF 1554TGCGGAACGTGGCAGAGCAGGCTGAGGTGGGTGTTC  819 CGTWQSRLGWVF 1555TGCGGAACGTGGCAGAGCAGGCTGGGGTGGGTGTTC  820 CGTWPRSLSAVWVF 1556TGCGGAACATGGCCTAGGAGCCTGAGTGCTGTTTGGGTGTTC  821 CGTWNNYLSAGDVVF 1557TGCGGAACATGGAATAACTACCTGAGTGCTGGCGATGTGGTTTTC  822 CGTWLGSQSPYWVF 1558TGCGGAACATGGCTTGGCAGCCAGAGTCCTTATTGGGTCTTC  823 CGTWHTGLSAYVF 1559TGCGGAACATGGCATACCGGCCTGAGTGCTTATGTCTTC  824 CGTWHSTLSAGHWVF 1560TGCGGAACATGGCATAGTACCCTGAGTGCTGGCCATTGGGTGTTC  825 CGTWHSSLSTWVF 1561TGCGGAACATGGCATAGTAGCCTGAGTACTTGGGTGTTC  826 CGTWHSSLSAYVF 1562TGCGGAACATGGCATAGCAGCCTGAGTGCCTATGTCTTC  827 CGTWHSSLSAVVF 1563TGCGGAACATGGCATAGCAGCCTGAGTGCTGTGGTATTC  828 CGTWHSGLSGWVF 1564TGCGGAACGTGGCATTCCGGCCTGAGTGGGTGGGTTTTC  829 CGTWHNTLRNVIF 1565TGCGGAACATGGCATAACACCCTGCGTAATGTGATATTC  830 CGTWHASLTAVF 1566TGCGGAACATGGCATGCCAGCCTGACTGCTGTGTTC  831 CGTWGWYGSQRGVVF 1567TGCGGGACATGGGGATGGTATGGCAGCCAGAGAGGCGTCGTCTTC  832 CGTWGWYGGQRGVVF 1568TGCGGGACATGGGGATGGTATGGCGGCCAGAGAGGCGTCGTCTTC  833 CGTWGTSLSAWVF 1569TGCGGAACCTGGGGAACCAGCCTGAGTGCTTGGGTGTTC  834 CGTWGSSLTTGLF 1570TGCGGAACCTGGGGTAGCAGCCTGACTACTGGCCTGTTC  835 CGTWGSSLTAYVF 1571TGCGGAACATGGGGTAGCAGCCTGACTGCCTATGTCTTC  836 CGTWGSSLSVVF 1572TGCGGAACATGGGGTAGCAGCCTGAGTGTTGTGTTC  837 CGTWGSSLSGGVF 1573TGCGGAACATGGGGTAGCAGCCTGAGTGGTGGGGTGTTC  838 CGTWGSSLSAYWVF 1574TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATTGGGTGTTC  839 CGTWGSSLSAYVVF 1575TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTGGTGTTC  840 CGTWGSSLSAYVF 1576TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTCTTC  841 CGTWGSSLSAVVF 1577TGCGGAACGTGGGGTAGTAGCCTGAGTGCTGTGGTGTTC  842 CGTWGSSLSAPYVF 1578TGCGGAACATGGGGTAGCAGCCTGAGTGCTCCTTATGTCTTC  843 CGTWGSSLSAPVF 1579TGCGGAACATGGGGTAGCAGCCTGAGTGCCCCGGTGTTC  844 CGTWGSSLSAGVF 1580TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGTGTTC  845 CGTWGSSLSAGLF 1581TGCGGAACTTGGGGTAGCAGCCTGAGTGCTGGACTGTTC  846 CGTWGSSLSAGALF 1582TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGCACTCTTC  847 CGTWGSSLRAWVF 1583TGCGGAACATGGGGCAGTAGCCTGCGTGCTTGGGTGTTC  848 CGTWFTSLASGVF 1584TGCGGAACCTGGTTTACTAGTCTGGCTAGTGGGGTTITC  849 CGTWETSLSVVVI 1585TGCGGAACTTGGGAGACCAGTCTGAGTGTCGTGGTCATC  850 CGTWETSLSGVF 1586TGCGGAACATGGGAGACCAGCCTGAGTGGTGTCTTC  851 CGTWETSLSDWVF 1587TGCGGAACATGGGAAACCAGCCTGAGTGATTGGGTATTC  852 CGTWETSLSAGVF 1588TGCGGAACATGGGAGACCAGCCTGAGTGCTGGGGTATTC  853 CGTWETSLNYVAF 1589TGCGGAACATGGGAAACCAGCCTTAATTATGTGGCCTTC  854 CGTWETSLNTWLL 1590TGCGGAACATGGGAGACCAGCCTGAATACTTGGTTGCTC  855 CGTWETSESGNYIF 1591TGCGGAACATGGGAGACCAGCGAGAGTGGTAATTACATCTTC  856 CGTWETRLGTWVI 1592TGCGGAACATGGGAAACCAGACTGGGTACTTGGGTGATC  857 CGTWETQLYWVF 1593TGCGGAACATGGGAGACCCAGTTATATTGGGTGTTC  858 CGTWETGLSAGEVF 1594TGCGGAACATGGGAGACTGGCCTAAGTGCTGGAGAGGTGTTC  859 CGTWESTLSVFLF 1595TGCGGAACTTGGGAAAGCACCCTGAGTGTTTTCCTATTC  860 CGTWESSLTVVVF 1596TGCGGGACATGGGAAAGTAGCCTGACTGTTGTGGTCTTC  861 CGTWESSLTGVVF 1597TGCGGAACATGGGAAAGTAGCCTGACTGGAGTGGTATTC  862 CGTWESSLTGFVF 1598TGCGGAACATGGGAAAGCAGCCTGACTGGTTTTGTCTTC  863 CGTWESSLSVGVF 1599TGTGGAACATGGGAGAGCAGCCTGAGTGTTGGGGTGTTC  864 CGTWESSLSEWVF 1600TGCGGAACCTGGGAAAGTAGCCTCAGTGAATGGGTGTTC  865 CGTWESSLSAVF 1601TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTATTC  866 CGTWESSLSAGYIF 1602TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGTTATATCTTC  867 CGTWESSLSAGVF 1603TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGAGTGTTC  868 CGTWESSLSAGPVF 1604TGCGGAACATGGGAAAGCAGCCTGAGCGCTGGCCCGGTGTTC  869 CGTWESSLSAGGQVF 1605TGCGGAACATGGGAAAGCAGCCTGAGTGCTGGAGGCCAGGTGTTC  870 CGTWESSLSAFGGYVF 1606TGCGGAACATGGGAGAGCAGCCTGAGTGCCTTCGGCGGTTATGTC TTC  871 CGTWESSLRVWVF1607 TGCGGAACATGGGAAAGCAGCCTGAGGGTTTGGGTGTTC  872 CGTWESSLFTGPWVF 1608TGCGGAACATGGGAAAGCAGCCTCTTTACTGGGCCTTGGGTGTTC  873 CGTWESLSATYVF 1609TGCGGAACATGGGAGAGCCTGAGTGCCACCTATGTCTTC  874 CGTWESGLSAGVF 1610TGCGGAACATGGGAGAGCGGCCTGAGTGCTGGTGTCTTC  875 CGTWESDFWVF 1611TGCGGAACATGGGAAAGCGACTTTTGGGTGTTT  876 CGTWENRLSAVVF 1612TGCGGTACATGGGAAAACAGACTGAGTGCTGTGGTCTTC  877 CGTWENRLSAGVF 1613TGCGGAACATGGGAAAACAGACTGAGTGCCGGGGTATTC  878 CGTWEISLTTSVVF 1614TGCGGAACATGGGAAATCAGCCTGACTACTTCTGTGGTATTC  879 CGTWEISLSTSVVF 1615TGCGGAACATGGGAAATCAGCCTGAGTACTTCTGTGGTATTC  880 CGTWEGSLSVVF 1616TGCGGAACATGGGAAGGCAGCCTCAGTGTTGTTTTC  881 CGTWEGSLRVF 1617TGCGGAACATGGGAAGGCAGCCTGAGGGTGTTC  882 CGTWEGSLRHVF 1618TGCGGAACATGGGAGGGCAGCCTGAGGCACGTGTTC  883 CGTWDYSPVRAGVF 1619TGCGGAACATGGGATTACAGCCCTGTACGTGCTGGGGTGTTC  884 CGTWDYSLSVYLF 1620TGCGGAACGTGGGATTACAGCCTGAGTGTTTATCTCTTC  885 CGTWDYSLSSGVVF 1621TGCGGAACATGGGATTACAGCCTGAGTTCTGGCGTGGTATTC  886 CGTWDYSLSAWVF 1622TGCGGAACATGGGATTACAGCCTGAGTGCCTGGGTGTTC  887 CGTWDYSLSAEVF 1623TGCGGAACATGGGATTACAGTCTGAGTGCTGAGGTGTTC  888 CGTWDYSLRRAIF 1624TGCGGAACATGGGATTACAGCCTGCGTCGTGCGATATTC  889 CGTWDWSLILQLF 1625TGCGGAACATGGGATTGGAGCCTCATTCTTCAATTGTTC  890 CGTWDVTLHTGVF 1626TGCGGAACATGGGATGTCACCTTGCATACTGGGGTGTTC  891 CGTWDVTLHIGVF 1627TGCGGAACATGGGATGTCACCTTGCATATTGGGGTGTTC  892 CGTWDVTLHAGVF 1628TGCGGAACATGGGATGTCACCTTGCATGCTGGGGTGTTC  893 CGTWDVSLYSGGVF 1629TGCGGAACATGGGATGTCAGTTTGTATAGTGGCGGGGTCTTC  894 CGTWDVSLTSFVF 1630TGTGGAACATGGGATGTCAGCCTGACTTCTTTCGTCTTC  895 CGTWDVSLSVGVL 1631TGCGGAACATGGGATGTCAGCCTGAGTGTTGGGGTGCTC  896 CGTWDVSLSAGDVVF 1632TGCGGAACGTGGGATGTCAGCCTGAGTGCTGGCGATGTAGTTTTC  897 CGTWDVSLNVVVF 1633TGCGGAACATGGGATGTCAGCCTGAATGTCGTGGTTTTC  898 CGTWDVSLNTQVF 1634TGCGGAACATGGGATGTCAGCCTGAATACTCAGGTGTTC  899 CGTWDVSLGALF 1635TGCGGCACATGGGATGTGAGCCTGGGTGCGCTGTTC  900 CGTWDVNLKTVVF 1636TGCGGAACGTGGGACGTTAATCTGAAAACTGTCGTTTTC  901 CGTWDVILSAEVF 1637TGCGGAACATGGGATGTCATCCTGAGTGCTGAGGTATTC  902 CGTWDTTVSAVVF 1638TGCGGAACATGGGATACCACCGTGAGTGCTGTGGTTTTC  903 CGTWDTTLTAWVF 1639TGCGGAACATGGGATACCACCCTGACTGCCTGGGTGTTC  904 CGTWDTTLSVFLF 1640TGCGGAACATGGGACACCACCTTGAGTGTTTTCCTATTC  905 CGTWDTSVSAGVF 1641TGCGGGACTTGGGATACCAGTGTGAGTGCTGGGGTGTTC  906 CGTWDTSVISWVF 1642TGCGGAACATGGGATACCAGTGTGATTTCTTGGGTTTTC  907 CGTWDTSRSSLYVVF 1643TGCGGAACATGGGATACCAGTCGGAGTTCTCTCTATGTGGTCTTC  908 CGTWDTSRSAWVF 1644TGCGGAACATGGGATACCAGCCGGAGTGCTTGGGTATTC  909 CGTWDTSRNPGGIF 1645TGCGGAACATGGGATACCAGCCGGAATCCTGGAGGAATTTTC  910 CGTWDTSRGHVF 1646TGCGGAACATGGGACACCAGTCGGGGTCATGTTTTC  911 CGTWDTSPSTGQVLF 1647TGCGGAACATGGGATACCAGCCCGAGTACTGGCCAGGTGCTTTTC  912 CGTWDTSPSAWVF 1648TGCGGAACATGGGATACCAGCCCGAGTGCCTGGGTGTTC  913 CGTWDTSLTWVF 1649TGCGGAACATGGGATACTAGCCTGACCTGGGTGTTC  914 CGTWDTSLTWFAVF 1650TGCGGAACATGGGATACCAGCCTGACGTGGTTCGCAGTGTTC  915 CGTWDTSLTVVVF 1651TGCGGAACATGGGATACCAGCCTGACTGTTGTGGTATTC  916 CGTWDTSLTTSWVF 1652TGCGGAACATGGGATACCAGCCTGACTACTTCTTGGGTGTTC  917 CGTWDTSLTTGPFWVF 1653TGCGGAACATGGGATACCAGCCTGACCACTGGTCCTTTTTGGGTGT TC  918 CGTWDTSLTPFYVF1654 TGCGGAACATGGGATACCAGCCTGACTCCTTTTTATGTCTTC  919 CGTWDTSLTAYVF 1655TGCGGAACATGGGATACCAGCCTGACTGCTTATGTCTTC  920 CGTWDTSLTAWVF 1656TGCGGAACATGGGATACCAGCCTGACTGCTTGGGTGTTC  921 CGTWDTSLTAWGVF 1657TGCGGAACATGGGATACCAGCCTGACTGCGTGGGGGGTGTTC  922 CGTWDTSLTAVVL 1658TGCGGCACATGGGATACCAGCCTGACTGCGGTGGTTCTC  923 CGTWDTSLTARVF 1659TGCGGAACCTGGGATACCAGCCTGACTGCTCGGGTTTTC  924 CGTWDTSLTAIVF 1660TGCGGAACATGGGATACCAGCCTGACTGCGATTGTCTTC  925 CGTWDTSLTAGVF 1661TGCGGAACATGGGATACCAGCCTGACTGCTGGTGTCTTC  926 CGTWDTSLSVYVF 1662TGCGGAACATGGGATACCAGCCTGAGTGTTTATGTCTTC  927 CGTWDTSLSVVF 1663TGCGGAACATGGGATACCAGCCTGAGTGTGGTGTTC  928 CGTWDTSLSVGEF 1664TGCGGGACATGGGATACCAGCCTGAGTGTTGGGGAATTC  929 CGTWDTSLSTWVF 1665TGCGGAACATGGGATACCAGCCTGAGTACTTGGGTGTTC  930 CGTWDTSLSTVVF 1666TGCGGAACATGGGATACCAGCCTGAGTACTGTGGTATTC  931 CGTWDTSLSTGQVLF 1667TGCGGAACATGGGATACCAGCCTGAGTACTGGCCAGGTGCTTTTC  932 CGTWDTSLSTGPLWVF 1668TGCGGCACATGGGATACCAGCCTGAGCACTGGTCCTCTTTGGGTGT TC  933 CGTWDTSLSSYVF1669 TGCGGAACTTGGGATACCAGCCTGAGTTCTTATGTCTTC  934 CGTWDTSLSSVVF 1670TGCGGAACATGGGATACCAGCCTGAGTTCTGTGGTCTTC  935 CGTWDTSLSSRYIF 1671TGCGGAACATGGGATACCAGCCTGAGTTCTAGATACATATTC  936 CGTWDTSLSSRFIF 1672TGCGGAACATGGGATACCAGCCTGAGTTCTAGATTCATATTC  937 CGTWDTSLSSGWVF 1673TGCGGAACATGGGATACCAGCCTGAGTTCTGGGTGGGTGTTC  938 CGTWDTSLSRYVF 1674TGCGGAACATGGGATACCAGCCTGAGTCGGTATGTGTTC  939 CGTWDTSLSQWLF 1675TGCGGAACTTGGGATACCAGTCTGAGTCAATGGCTGTTC  940 CGTWDTSLSPGLWVF 1676TGCGGAACATGGGATACCAGCCTGAGTCCTGGCCTTTGGGTGTTC  941 CGTWDTSLSNYVF 1677TGCGGAACATGGGATACCAGCCTGAGTAATTATGTCTTC  942 CGTWDTSLSIWVF 1678TGCGGAACATGGGATACCAGCCTAAGTATTTGGGTGTTC  943 CGTWDTSLSIGPFWVF 1679TGCGGCACATGGGATACCAGCCTGAGCATTGGTCCTTTTTGGGTGT TC  944 CGTWDTSLSGWVF1680 TGCGGAACATGGGATACCAGCCTGAGTGGTTGGGTGTTC  945 CGTWDTSLSGTVF 1681TGCGGAACATGGGATACCAGCCTGAGTGGTACAGTGTTC  946 CGTWDTSLSGGQVF 1682TGCGGAACATGGGATACTAGTCTGAGTGGTGGCCAGGTGTTC  947 CGTWDTSLSGGIF 1683TGCGGAACATGGGATACCAGCCTGAGTGGTGGGATATTC  948 CGTWDTSLSGEDVVI 1684TGCGGAACATGGGATACCAGCCTGAGTGGTGAGGATGTGGTAATC  949 CGTWDTSLSFLYAF 1685TGCGGAACATGGGATACCAGCCTGAGTTTCCTTTATGCTTTC  950 CGTWDTSLSEVVF 1686TGCGGAACATGGGATACCAGCCTGAGTGAGGTCGTATTC  951 CGTWDTSLSEVF 1687TGCGGAACATGGGATACCAGCCTGAGTGAAGTGTTC  952 CGTWDTSLSENWVF 1688TGCGGAACATGGGATACTAGCCTGAGTGAAAATTGGGTGTTC  953 CGTWDTSLSAYIF 1689TGCGGAACATGGGATACCAGCCTGAGTGCCTACATATTC  954 CGTWDTSLSAVVL 1690TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTACTC  955 CGTWDTSLSAVF 1691TGCGGAACATGGGATACCAGCCTGAGTGCTGTTTTC  956 CGTWDTSLSARVF 1692TGCGGAACATGGGATACCAGCCTGAGTGCCCGGGTGTTC  957 CGTWDTSLSARQVF 1693TGCGGCACATGGGATACCAGCCTGAGTGCCCGCCAGGTATTC  958 CGTWDTSLSALVF 1694TGCGGAACATGGGATACCAGCCTGAGTGCTTTGGTTTTC  959 CGTWDTSLSAKVF 1695TGCGGAACATGGGATACCAGCCTGAGTGCTAAGGTGTTC  960 CGTWDTSLSAKIF 1696TGCGGAACATGGGATACCAGCCTGAGTGCGAAAATCTTC  961 CGTWDTSLSAKAVF 1697TGCGGAACATGGGATACCAGCCTGAGTGCCAAGGCGGTATTC  962 CGTWDTSLSAHAVF 1698TGCGGAACATGGGATACCAGCCTGAGTGCCCATGCTGTGTTC  963 CGTWDTSLSAGYVF 1699TGCGGAACATGGGATACCAGCCTGAGTGCTGGCTATGTCTTC  964 CGTWDTSLSAGRWVF 1700TGCGGAACATGGGACACCAGTCTGAGTGCTGGCCGCTGGGTGTTC  965 CGTWDTSLSAGIF 1701TGCGGAACATGGGATACCAGCCTGAGTGCTGGGATATTC  966 CGTWDTSLSAGGFRVF 1702TGCGGAACATGGGATACCAGCCTGAGTGCTGGTGGGTTCCGGGTC TTC  967 CGTWDTSLSAGAF1703 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGCATTC  968 CGTWDTSLSADWFF 1704TGCGGAACATGGGATACCAGTCTGAGTGCTGATTGGTTTTTC  969 CGTWDTSLSADEYVF 1705TGCGGAACATGGGATACCAGCCTGAGTGCTGATGAATATGTCTTC  970 CGTWDTSLSAAWVF 1706TGCGGCACATGGGATACCAGCCTGAGTGCGGCTTGGGTGTTC  971 CGTWDTSLSAALF 1707TGCGGAACATGGGATACCAGCCTGAGTGCTGCGCTATTC  972 CGTWDTSLSAAGVF 1708TGCGGAACATGGGATACCAGCCTGAGTGCTGCGGGGGTTTTC  973 CGTWDTSLRVVVF 1709TGCGGAACATGGGATACCAGCCTGAGAGTTGTGGTTTTC  974 CGTWDTSLRTWVF 1710TGCGGAACATGGGATACCAGCCTGAGAACCTGGGTATTC  975 CGTWDTSLRGAVF 1711TGCGGAACGTGGGATACCAGCCTGAGGGGTGCAGTGTTC  976 CGTWDTSLRAVVF 1712TGCGGAACATGGGATACCAGCCTGCGTGCTGTGGTATTC  977 CGTWDTSLNVVYVF 1713TGCGGAACATGGGATACAAGCCTGAATGTAGTTTATGTCTTC  978 CGTWDTSLNTYLF 1714TGCGGAACATGGGATACCAGCCTCAACACCTACCTGTTC  979 CGTWDTSLNFAWLF 1715TGCGGAACATGGGATACTAGCCTGAACTTCGCTTGGCTGTTC  980 CGTWDTSLLVWLF 1716TGCGGCACATGGGATACCAGCCTTCTTGTGTGGCTTTTC  981 CGTWDTSLKTWVF 1717TGCGGAACATGGGATACCAGTCTGAAGACGTGGGTGTTC  982 CGTWDTSLIVWVF 1718TGCGGAACATGGGATACCAGTCTGATTGTCTGGGTGTTC  983 CGTWDTSLITGVF 1719TGCGGAACATGGGATACCAGCCTAATTACTGGGGTGTTC  984 CGTWDTSLISVVF 1720TGCGGAACATGGGATACCAGCCTGATTAGCGTGGTATTC  985 CGTWDTSLIAYVF 1721TGCGGAACATGGGATACCAGCCTGATTGCTTATGTCTTC  986 CGTWDTSLHTELF 1722TGCGGAACATGGGATACCAGCCTGCACACTGAGTTGTTC  987 CGTWDTSLGSYVF 1723TGCGGAACTTGGGATACCAGCCTGGGTTCTTATGTCTTC  988 CGTWDTSLGSLWVF 1724TGCGGAACATGGGATACCAGCCTGGGTTCTCTTTGGGTGTTC  989 CGTWDTSLGSGVF 1725TGCGGTACATGGGATACCAGCCTGGGTTCTGGGGTATTC  990 CGTWDTSLGGRGVF 1726TGCGGAACTTGGGATACCAGTCTGGGTGGTAGAGGGGTCTTC  991 CGTWDTSLGAWVF 1727TGCGGAACATGGGATACCAGCCTGGGTGCTTGGGTGTTC  992 CGTWDTSLGAVVF 1728TGCGGAACATGGGATACCAGCCTGGGTGCCGTGGTATTC  993 CGTWDTSLGAGVF 1729TGCGGAACATGGGATACCAGCCTGGGTGCTGGGGTATTC  994 CGTWDTSLGAGLF 1730TGCGGAACATGGGATACCAGCCTGGGTGCTGGCCTATTC  995 CGTWDTSLDAVVF 1731TGCGGAACATGGGATACCAGTCTGGATGCTGTGGTTTTC  996 CGTWDTSLDAVLF 1732TGCGGGACTTGGGATACCAGCCTGGATGCTGTGCTGTTC  997 CGTWDTSLAWVF 1733TGCGGAACATGGGATACCAGCCTGGCTTGGGTGTTC  998 CGTWDTSLATGLF 1734TGCGGAACATGGGATACCAGCCTGGCGACTGGACTGTTC  999 CGTWDTSLAPVVF 1735TGCGGGACATGGGATACCAGCCTGGCCCCTGTAGTCTTC 1000 CGTWDTRLTIVIF 1736TGCGGAACATGGGACACCCGCCTGACTATTGTGATCTTC 1001 CGTWDTRLSVWLF 1737TGTGGAACATGGGACACCAGGCTGAGTGTTTGGCTGTTC 1002 CGTWDTRLSVGVF 1738TGCGGAACGTGGGACACCAGACTGAGTGTTGGGGTTTTC 1003 CGTWDTRLSTVIF 1739TGCGGCACATGGGATACCAGACTGAGTACTGTAATTTTC 1004 CGTWDTRLSSVVF 1740TGCGGAACATGGGATACCCGCCTGAGTTCTGTGGTCTTC 1005 CGTWDTRLSIVVF 1741TGCGGAACATGGGATACCCGCCTGAGTATTGTGGTTTTC 1006 CGTWDTRLSAYVVF 1742TGCGGAACATGGGATACCAGACTGAGTGCCTATGTGGTATTC 1007 CGTWDTRLSAWVF 1743TGCGGAACCTGGGACACCCGCCTGAGTGCGTGGGTGTTC 1008 CGTWDTRLSAVVF 1744TGCGGAACATGGGATACCAGACTGAGTGCTGTGGTGTTC 1009 CGTWDTRLSAGLF 1745TGCGGAACATGGGATACCCGCCTGAGTGCTGGGTTGTTC 1010 CGTWDTRLSAGGVF 1746TGCGGAACATGGGATACCAGACTGAGTGCTGGTGGGGTGTTC 1011 CGTWDTRLNVWLF 1747TGCGGAACATGGGATACCAGATTGAATGTGTGGCTATTC 1012 CGTWDTNREVVLL 1748TGCGGAACATGGGATACCAACCGGGAAGTTGTGCTCCTC 1013 CGTWDTNLRAHVF 1749TGCGGAACATGGGATACCAACCTGCGTGCCCATGTCTTC 1014 CGTWDTNLPAVVF 1750TGCGGAACATGGGATACTAATCTGCCCGCTGTAGTGTTC 1015 CGTWDTNLGGVF 1751TGCGGAACATGGGACACCAATTTGGGTGGGGTGTTC 1016 CGTWDTIVSIGVF 1752TGCGGAACATGGGATACCATCGTGAGTATTGGGGTGTTC 1017 CGTWDTILSAVVF 1753TGCGGAACATGGGATACCATCCTGAGTGCGGTGGTGTTC 1018 CGTWDTILSAEVF 1754TGCGGCACATGGGATACCATCCTGAGTGCTGAGGTGTTC 1019 CGTWDTHLGVVF 1755TGCGGAACATGGGATACCCACCTGGGTGTGGTTTTC 1020 CGTWDTGPSPHWLF 1756TGCGGAACATGGGATACCGGCCCGAGCCCTCATTGGCTGTTC 1021 CGTWDTGLTFGGVF 1757TGCGGAACATGGGATACCGGCCTGACTTTTGGAGGCGTGTTC 1022 CGTWDTGLTAFVF 1758TGCGGAACATGGGATACCGGCCTGACTGCTTTTGTCTTC 1023 CGTWDTGLSVWVF 1759TGCGGAACATGGGATACCGGCCTGAGTGTTTGGGTGTTC 1024 CGTWDTGLSTGIF 1760TGCGGAACATGGGATACCGGCCTGAGTACTGGGATTTTC 1025 CGTWDTGLSSLLF 1761TGCGGAACATGGGATACCGGCCTGAGTTCCCTGCTCTTC 1026 CGTWDTGLSIVVF 1762TGCGGAACGTGGGACACCGGCCTGAGTATTGTGGTGTTC 1027 CGTWDTGLSFVVF 1763TGCGGAACGTGGGACACCGGCCTGAGTTTTGTGGTGTTC 1028 CGTWDTGLSAWVF 1764TGCGGAACATGGGATACCGGCCTGAGTGCTTGGGTGTTC 1029 CGTWDTGLSAGVVF 1765TGCGGAACATGGGATACCGGCCTGAGTGCTGGTGTGGTATTC 1030 CGTWDTGLRGWIF 1766TGCGGAACATGGGATACCGGTCTGAGGGGTTGGATTTTC 1031 CGTWDTELSAGVF 1767TGCGGAACATGGGATACCGAGCTAAGTGCGGGGGTCTTC 1032 CGTWDTALTAGVF 1768TGCGGAACGTGGGATACCGCCCTGACTGCTGGGGTGTTC 1033 CGTWDTALSLVVF 1769TGCGGAACATGGGATACTGCCCTGAGTCTTGTGGTCTTC 1034 CGTWDTALSAWLF 1770TGCGGAACATGGGATACCGCCCTGAGTGCCTGGCTGTTC 1035 CGTWDTALSAGVF 1771TGCGGCACATGGGATACCGCCCTGAGTGCTGGGGTGTTC 1036 CGTWDTALRGVLF 1772TGCGGAACATGGGATACCGCCCTGCGTGGCGTGCTGTTC 1037 CGTWDTALKEWLF 1773TGCGGAACATGGGATACCGCCCTGAAAGAATGGCTGTTC 1038 CGTWDRTLTAGDVLF 1774TGCGGAACATGGGATAGGACCCTGACTGCTGGCGATGTGCTCTTC 1039 CGTWDRSVTYVF 1775TGCGGAACATGGGATAGAAGCGTGACTTATGTCTTC 1040 CGTWDRSRNEWVF 1776TGCGGAACATGGGATCGCAGCCGAAATGAATGGGTGTTC 1041 CGTWDRSLTVWVF 1777TGCGGAACATGGGATCGCAGTCTGACTGTTTGGGTCTTC 1042 CGTWDRSLTPGWLF 1778TGCGGAACATGGGATCGCAGCCTGACTCCTGGGTGGTTGTTC 1043 CGTWDRSLTAWVF 1779TGCGGAACATGGGATAGAAGCCTGACTGCTTGGGTGTTC 1044 CGTWDRSLSVVVF 1780TGCGGAACATGGGACCGCAGCCTGAGTGTTGTGGTATTC 1045 CGTWDRSLSVVF 1781TGCGGCACATGGGATCGCAGCCTGAGTGTAGTCTTC 1046 CGTWDRSLSVQLF 1782TGCGGAACATGGGATAGGAGCCTGAGTGTTCAATTGTTC 1047 CGTWDRSLSVLWVF 1783TGCGGAACATGGGATCGCAGCCTCAGTGTTCTTTGGGTGTTC 1048 CGTWDRSLSVGLF 1784TGCGGAACATGGGATCGCAGCCTGAGTGTTGGATTATTC 1049 CGTWDRSLSTWVF 1785TGCGGAACATGGGATCGCAGCCTGAGTACTTGGGTGTTC 1050 CGTWDRSLSTHWVL 1786TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGGGTGCTC 1051 CGTWDRSLSTHWVF 1787TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGGGTGTTC 1052 CGTWDRSLSSAVF 1788TGCGGAACCTGGGATCGAAGCCTGAGTTCTGCGGTGTTC 1053 CGTWDRSLSPSYVF 1789TGCGGAACATGGGACAGAAGCCTGAGTCCCTCTTATGTCTTC 1054 CGTWDRSLSGEVF 1790TGCGGAACATGGGATAGGAGCCTGAGTGGTGAGGTGTTC 1055 CGTWDRSLSGAVF 1791TGCGGAACATGGGATAGGAGCCTGAGTGGTGCGGTGTTC 1056 CGTWDRSLSAVAF 1792TGCGGAACATGGGATCGCAGCCTGAGTGCTGTGGCATTC 1057 CGTWDRSLSAGGEF 1793TGCGGAACATGGGATAGGAGCCTGAGTGCCGGGGGGGAATTC 1058 CGTWDRSLSAFWVF 1794TGCGGAACATGGGATCGCAGCCTGAGTGCTTTTTGGGTGTTC 1059 CGTWDRSLSAAVF 1795TGCGGAACATGGGATAGGAGCCTGAGTGCTGCGGTGTTC 1060 CGTWDRSLSAALF 1796TGCGGAACATGGGATAGGAGCCTGAGTGCTGCACTCTTC 1061 CGTWDRSLRVF 1797TGCGGAACATGGGATCGCAGCCTGAGAGTGTTC 1062 CGTWDRSLNWVF 1798TGCGGTACATGGGACAGAAGCCTTAATTGGGTGTTC 1063 CGTWDRSLNVYVF 1799TGCGGAACATGGGATCGCAGCCTGAATGTTTATGTCTTC 1064 CGTWDRSLNVGVF 1800TGCGGAACATGGGATAGGAGCCTGAATGTTGGGGTGTTC 1065 CGTWDRSLHVVF 1801TGCGGAACATGGGATCGGAGCCTGCATGTGGTCTTC 1066 CGTWDRSLGGWVF 1802TGTGGAACATGGGATCGCAGCCTGGGTGGTTGGGTGTTC 1067 CGTWDRSLGAFWVF 1803TGCGGAACATGGGATCGCAGCCTGGGTGCTTTTTGGGTGTTC 1068 CGTWDRSLFWVF 1804TGCGGAACATGGGATAGAAGCCTGTTTTGGGTGTTC 1069 CGTWDRSLAAGVF 1805TGCGGAACGTGGGATCGCAGCCTGGCTGCTGGGGTGTTC 1070 CGTWDRRLSGVVF 1806TGCGGAACATGGGATAGGAGGTTGAGTGGTGTCGTATTC 1071 CGTWDRRLSDVVF 1807TGCGGAACGTGGGATCGCCGCCTAAGTGATGTGGTATTC 1072 CGTWDRRLSAVVF 1808TGCGGAACATGGGATAGGAGGCTGAGTGCTGTGGTATTC 1073 CGTWDRRLNVAFF 1809TGCGGAACATGGGATAGACGCCTGAATGTTGCGTTCTTC 1074 CGTWDRRLLAVF 1810TGTGGAACATGGGATAGGAGGCTGCTTGCTGTTTTC 1075 CGTWDRNLRAVVF 1811TGCGGAACTTGGGATAGGAACCTGCGCGCCGTGGTCTTC 1076 CGTWDRLSAGVF 1812TGCGGAACATGGGATAGGCTGAGTGCTGGGGTGTTC 1077 CGTWDRGPNTGVF 1813TGCGGAACATGGGATAGAGGCCCGAATACTGGGGTATTC 1078 CGTWDRGLNTVYVF 1814TGCGGAACATGGGATAGAGGCCTGAATACTGTTTACGTCTTC 1079 CGTWDNYVSAPWVF 1815TGCGGAACATGGGATAACTATGTGAGTGCCCCTTGGGTGTTC 1080 CGTWDNYLSAGDVVF 1816TGCGGAACATGGGATAACTACCTGAGTGCTGGCGATGTGGTTTTC 1081 CGTWDNYLRAGVF 1817TGCGGAACATGGGATAACTACCTGAGAGCTGGGGTCTTC 1082 CGTWDNYLGAVVF 1818TGCGGAACATGGGACAATTATCTGGGTGCCGTGGTTTTC 1083 CGTWDNYLGAGVF 1819TGCGGAACATGGGATAACTACCTGGGTGCGGGGGTGTTC 1084 CGTWDNTVSAPWVF 1820TGCGGAACATGGGATAACACCGTGAGTGCCCCTTGGGTTTTC 1085 CGTWDNTLSLWVF 1821TGCGGAACATGGGATAACACCCTGAGTCTTTGGGTGTTC 1086 CGTWDNTLSAGVF 1822TGCGGAACATGGGATAACACCCTGAGTGCTGGGGTCTTC 1087 CGTWDNTLLTVLF 1823TGCGGAACATGGGACAACACTCTGCTTACTGTGTTATTC 1088 CGTWDNRLSSVIF 1824TGCGGAACATGGGATAACAGACTGAGTAGTGTGATTTTC 1089 CGTWDNRLSAVVF 1825TGCGGAACATGGGATAACAGGTTGAGTGCTGTGGTCTTC 1090 CGTWDNRLSAGGIF 1826TGCGGAACATGGGATAACAGGCTGAGTGCTGGTGGGATATTC 1091 CGTWDNRLSAEVF 1827TGCGGAACATGGGATAACAGACTGAGTGCTGAGGTGTTC 1092 CGTWDNRLRVGVL 1828TGTGGAACATGGGATAACAGACTGCGTGTTGGGGTTCTC 1093 CGTWDNRLLENVF 1829TGCGGAACATGGGATAATCGCCTGCTTGAGAATGTCTTC 1094 CGTWDNNLRAVF 1830TGCGGAACATGGGATAACAACCTGCGTGCTGTCTTC 1095 CGTWDNNLRAGVF 1831TGCGGAACTTGGGATAATAACCTGCGTGCTGGAGTGTTC 1096 CGTWDNNLGGGRVF 1832TGCGGAACATGGGACAACAATTTGGGCGGTGGCCGGGTGTTC 1097 CGTWDNNLGAGVL 1833TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTCCTC 1098 CGTWDNNLGAGVF 1834TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTCTTC 1099 CGTWDNILSAAVF 1835TGCGGAACTTGGGATAACATCCTGAGCGCTGCGGTGTTC 1100 CGTWDNILDAGVF 1836TGCGGAACCTGGGATAACATCTTGGATGCAGGGGTTTTC 1101 CGTWDNDLSGWLF 1837TGCGGAACATGGGATAACGACCTGAGTGGTTGGCTGTTC 1102 CGTWDNDLSAWVF 1838TGCGGAACATGGGATAACGACCTGAGTGCCTGGGTGTTC 1103 CGTWDLTLGGVVF 1839TGCGGAACATGGGATCTCACCCTGGGTGGTGTGGTGTTC 1104 CGTWDLSLSAGVF 1840TGCGGAACATGGGATCTCAGCCTGAGTGCTGGGGTATTC 1105 CGTWDLSLKEWVF 1841TGCGGAACATGGGATCTCAGCCTGAAAGAATGGGTGTTC 1106 CGTWDLSLDAVVF 1842TGCGGAACGTGGGATCTCAGCCTGGATGCTGTTGTTTTC 1107 CGTWDLKVF 1843TGCGGAACCTGGGACCTGAAGGTTTTC 1108 CGTWDKTLSVWVF 1844TGCGGAACATGGGATAAGACTCTGAGTGTTTGGGTGTTC 1109 CGTWDKSLSVWVF 1845TGCGGAACATGGGATAAGAGCCTGAGTGTTTGGGTGTTC 1110 CGTWDKSLSGVVF 1846TGCGGAACATGGGATAAGAGCCTGAGTGGTGTGGTATTT 1111 CGTWDKSLSDWVF 1847TGCGGAACATGGGATAAGAGCCTGAGTGATTGGGTGTTC 1112 CGTWDKSLSALVF 1848TGCGGAACATGGGATAAGAGCCTGAGTGCTTTGGTTTTC 1113 CGTWDKSLSAGVF 1849TGCGGAACATGGGATAAGAGCCTGAGTGCTGGCGTCTTC 1114 CGTWDKSLSADVF 1850TGCGGAACATGGGATAAGAGCCTGAGTGCCGACGTCTTC 1115 CGTWDKRLTIVVF 1851TGCGGAACATGGGATAAACGCCTGACTATTGTGGTCTTC 1116 CGTWDKRLSAWVL 1852TGCGGAACATGGGATAAACGCCTGAGTGCCTGGGTGCTC 1117 CGTWDKNLRAVVF 1853TGCGGAACATGGGATAAGAACCTGCGTGCTGTGGTCTTC 1118 CGTWDITLSGFVF 1854TGCGGAACATGGGATATCACCCTGAGTGGGTTTGTCTTC 1119 CGTWDITLHTGVF 1855TGCGGAACATGGGATATCACCTTGCATACTGGAGTATTC 1120 CGTWDISVTVVF 1856TGCGGAACATGGGATATCAGTGTGACTGTGGTGTTC 1121 CGTWDISVRGYAF 1857TGCGGAACATGGGATATCAGTGTGAGGGGTTATGCCTTC 1122 CGTWDISRWVF 1858TGCGGAACATGGGATATCAGCCGTTGGGTTTTC 1123 CGTWDISPSAWVF 1859TGCGGAACATGGGATATCAGCCCGAGTGCTTGGGTGTTC 1124 CGTWDISLSVWVF 1860TGCGGAACATGGGATATTAGCCTGAGTGTCTGGGTGTTC 1125 CGTWDISLSVVF 1861TGCGGAACATGGGATATCAGCCTGAGTGTTGTATTC 1126 CGTWDISLSSVVF 1862TGCGGAACTTGGGATATCAGCCTGAGTTCTGTGGTGTTC 1127 CGTWDISLSHWLF 1863TGCGGAACATGGGATATCAGCCTGAGTCACTGGTTGTTC 1128 CGTWDISLSGWVF 1864TGCGGAACATGGGATATCAGTCTGAGTGGTTGGGTGTTC 1129 CGTWDISLSGRVF 1865TGCGGAACATGGGATATCAGCCTGAGTGGTCGAGTGTTC 1130 CGTWDISLSAWAF 1866TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGCGTTC 1131 CGTWDISLSAVVF 1867TGCGGAACATGGGATATCAGCCTGAGTGCTGTGGTTTTC 1132 CGTWDISLSAVIF 1868TGCGGGACATGGGACATCAGCCTGAGTGCTGTGATATTC 1133 CGTWDISLSAVF 1869TGCGGAACATGGGATATCAGCCTGAGTGCTGTGTTC 1134 CGTWDISLSARVF 1870TGCGGAACATGGGATATCAGCCTGAGTGCCCGGGTGTTC 1135 CGTWDISLSALVF 1871TGCGGAACATGGGATATCAGCCTGAGTGCCCTGGTGTTC 1136 CGTWDISLSAHVF 1872TGCGGAACATGGGATATTAGCCTGAGTGCCCATGTCTTC 1137 CGTWDISLSAGVVF 1873TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTGGTATTC 1138 CGTWDISLSAGPYVF 1874TGCGGAACATGGGATATCAGCCTGAGTGCCGGCCCTTATGTCTTC 1139 CGTWDISLSAGGVF 1875TGCGGCACATGGGATATCAGCCTGAGTGCTGGAGGGGTGTTC 1140 CGTWDISLSAEVF 1876TGCGGAACATGGGATATCAGCCTGAGTGCTGAGGTTTTC 1141 CGTWDISLSAAVF 1877TGCGGAACATGGGATATCAGCCTGAGTGCTGCTGTGTTC 1142 CGTWDISLRAVF 1878TGCGGAACATGGGATATCAGCCTGCGTGCTGTGTTC 1143 CGTWDISLNTGVF 1879TGCGGAACATGGGATATTAGCCTGAATACTGGGGTGTTC 1144 CGTWDISLNNYVF 1880TGCGGAACATGGGATATCAGCCTAAATAATTATGTCTTC 1145 CGTWDISLIAGVF 1881TGCGGAACATGGGATATCAGCCTAATTGCTGGGGTATTC 1146 CGTWDISLHTWLF 1882TGCGGAACATGGGATATCAGCCTGCATACTTGGCTGTTC 1147 CGTWDIRLTDELLF 1883TGCGGAACATGGGATATCCGCCTGACCGATGAGCTGTTATTC 1148 CGTWDIRLSGFVF 1884TGCGGAACATGGGATATCAGACTGAGCGGTTTTGTTTTC 1149 CGTWDINLGAGGLYVF 1885TGCGGAACATGGGATATCAACCTGGGTGCTGGGGGCCTTTATGTC TTC 1150 CGTWDIILSAEVF1886 TGCGGAACATGGGATATCATCCTGAGTGCTGAGGTATTC 1151 CGTWDHTLSAVF 1887TGCGGAACATGGGATCACACCCTGAGTGCTGTCTTC 1152 CGTWDHTLLTVLF 1888TGCGGAACATGGGACCACACTCTGCTTACTGTGTTATTC 1153 CGTWDHSLTAVVF 1889TGCGGAACATGGGATCACAGCCTGACTGCTGTGGTATTC 1154 CGTWDHSLTAGIF 1890TGCGGAACCTGGGATCACAGCCTGACTGCTGGGATATTC 1155 CGTWDHSLSVVLF 1891TGCGGAACATGGGATCACAGCCTGAGTGTTGTATTATTC 1156 CGTWDHSLSLVF 1892TGCGGAACATGGGATCACAGCCTGAGTTTGGTATTC 1157 CGTWDHSLSIGVF 1893TGCGGAACATGGGATCACAGCCTGTCTATTGGGGTTTTC 1158 CGTWDHSLSAGVF 1894TGCGGAACATGGGATCACAGCCTGAGTGCTGGGGTGTTC 1159 CGTWDHSLSAFVF 1895TGTGGAACTTGGGATCACAGCCTGAGTGCTTTCGTGTTC 1160 CGTWDHSLSAAVF 1896TGCGGAACATGGGATCACAGTCTGAGTGCTGCTGTTTTC 1161 CGTWDHNLRAVF 1897TGCGGAACATGGGACCACAATCTGCGTGCTGTCTTC 1162 CGTWDFTLSVGRF 1898TGCGGGACATGGGATTTCACCCTGAGTGTTGGGCGCTTC 1163 CGTWDFTLSAPVF 1899TGCGGAACATGGGATTTCACCCTGAGTGCTCCTGTCTTC 1164 CGTWDFSVSAGWVF 1900TGCGGAACGTGGGATTTCAGCGTGAGTGCTGGGTGGGTGTTC 1165 CGTWDFSLTTWLF 1901TGCGGAACGTGGGATTTCAGTCTTACTACCTGGTTATTC 1166 CGTWDFSLSVWVF 1902TGCGGAACATGGGATTTCAGCCTGAGTGTTTGGGTGTTC 1167 CGTWDFSLSTGVF 1903TGCGGAACATGGGATTTCAGCCTGAGTACTGGGGTTTTC 1168 CGTWDFSLSGVVF 1904TGCGGCACATGGGATTTCAGCCTGAGTGGTGTGGTATTC 1169 CGTWDFSLSGFVF 1905TGCGGAACATGGGATTTCAGCCTGAGTGGTTTCGTGTTC 1170 CGTWDFSLSAGVF 1906TGCGGAACATGGGATTTCAGCCTGAGTGCTGGGGTGTTC 1171 CGTWDETVRGWVF 1907TGCGGAACATGGGATGAAACCGTGAGAGGTTGGGTGTTC 1172 CGTWDESLRSWVF 1908TGCGGAACATGGGATGAAAGTCTGAGAAGCTGGGTGTTC 1173 CGTWDERQTDESYVF 1909TGCGGAACTTGGGATGAGAGGCAGACTGATGAGTCCTATGTCTTC 1174 CGTWDERLVAGQVF 1910TGCGGAACATGGGATGAGAGACTCGTTGCTGGCCAGGTCTTC 1175 CGTWDERLSPGAFF 1911TGCGGAACATGGGATGAGAGACTGAGTCCTGGAGCTTTTTTC 1176 CGTWDEKVF 1912TGCGGAACATGGGATGAGAAGGTGTTC 1177 CGTWDEGQTTDFFVF 1913TGCGGAACCTGGGATGAAGGCCAGACTACTGATTTCTTTGTCTTC 1178 CGTWDDTLAGVVF 1914TGCGGAACATGGGATGACACCCTGGCTGGTGTGGTCTTC 1179 CGTWDDRLTSAVF 1915TGCGGAACATGGGATGACAGGCTGACTTCTGCGGTCTTC 1180 CGTWDDRLFVVVF 1916TGCGGAACATGGGATGACAGACTGTTTGTTGTGGTATTC 1181 CGTWDDNLRGWVF 1917TGCGGAACATGGGATGATAACCTGAGAGGTTGGGTGTTC 1182 CGTWDDNLRGVVF 1918TGCGGAACATGGGATGACAACCTGCGTGGTGTCGTGTTC 1183 CGTWDDNLNIGRVF 1919TGCGGAACCTGGGATGACAATTTGAATATTGGAAGGGTGTTC 1184 CGTWDDILSAVIF 1920TGCGGAACATGGGATGACATCCTGAGTGCTGTGATATTC 1185 CGTWDDILRGWVF 1921TGCGGAACATGGGATGATATCCTGAGAGGTTGGGTGTTC 1186 CGTWDATLSPGWLF 1922TGCGGAACATGGGATGCCACCCTGAGTCCTGGGTGGTTATTC 1187 CGTWDASVTSWVF 1923TGCGGAACATGGGATGCCAGCGTGACTTCTTGGGTGTTC 1188 CGTWDASLTSVVF 1924TGCGGAACATGGGATGCCAGCCTGACTTCTGTGGTCTTC 1189 CGTWDASLSVWVF 1925TGCGGAACATGGGATGCCAGCCTGAGTGTTTGGGTGTTC 1190 CGTWDASLSVPWVF 1926TGCGGAACATGGGATGCCAGCCTGAGTGTTCCTTGGGTGTTC 1191 CGTWDASLSVAVF 1927TGCGGAACATGGGATGCCAGCCTGAGTGTGGCGGTATTC 1192 CGTWDASLSTWVF 1928TGCGGAACATGGGATGCCAGCCTGAGTACCTGGGTATTC 1193 CGTWDASLSGVVF 1929TGCGGAACATGGGATGCCAGCCTGAGTGGTGTGGTATTC 1194 CGTWDASLSGGGEF 1930TGCGGAACATGGGATGCCAGCCTGAGTGGTGGGGGAGAATTC 1195 CGTWDASLSAGVF 1931TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGGTGTTC 1196 CGTWDASLSAGLF 1932TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGCTTTTC 1197 CGTWDASLSAEVF 1933TGTGGCACATGGGATGCCAGCCTGAGTGCTGAAGTCTTC 1198 CGTWDASLSADFWVF 1934TGCGGAACATGGGATGCCAGCCTGAGTGCTGACTTTTGGGTGTTC 1199 CGTWDASLRVFF 1935TGCGGAACATGGGATGCCAGCCTGAGAGTCTTCTTC 1200 CGTWDASLRAVVL 1936TGCGGAACATGGGATGCCAGTCTGAGGGCTGTGGTACTC 1201 CGTWDASLNIWVF 1937TGCGGAACATGGGATGCCAGCCTGAATATTTGGGTTTTC 1202 CGTWDASLKNLVF 1938TGCGGGACATGGGATGCCAGCCTGAAGAATCTGGTCTTC 2322 CGTWDASLGAWVF 1939TGCGGAACATGGGATGCCAGCCTGGGTGCCTGGGTATTC 2323 CGTWDASLGAVVF 1940TGCGGAACATGGGATGCCAGCCTGGGTGCTGTGGTCTTC 2324 CGTWDASLGAGVF 1941TGCGGAACATGGGATGCCAGCCTGGGTGCGGGGGTCTTC 2325 CGTWDARLSGLYVF 1942TGCGGAACATGGGATGCTAGGCTGAGTGGCCTTTATGTCTTC 2326 CGTWDARLGGAVF 1943TGTGGAACCTGGGATGCGAGACTGGGTGGTGCAGTCTTC 2327 CGTWDANLRAGVF 1944TGCGGAACATGGGATGCCAATCTGCGTGCTGGGGTCTTC 2328 CGTWDAIISGWVF 1945TGCGGAACATGGGATGCTATCATAAGTGGTTGGGTGTTC 2329 CGTWDAGQSVWVF 1946TGCGGAACATGGGATGCCGGCCAGAGTGTTTGGGTGTTC 2330 CGTWDAGLTGLYVF 1947TGCGGCACATGGGATGCCGGGCTGACTGGCCTTTATGTCTTC 2331 CGTWDAGLSVYVF 1948TGCGGAACTTGGGATGCCGGTCTGAGTGTTTATGTCTTC 2332 CGTWDAGLSTGVF 1949TGCGGGACATGGGATGCCGGCCTGAGTACTGGGGTCTTC 2333 CGTWDAGLSGDVF 1950TGCGGAACATGGGATGCCGGCCTGAGTGGGGACGTTTTC 2334 CGTWDAGLSAGYVF 1951TGCGGAACATGGGATGCCGGCCTGAGTGCTGGTTATGTCTTC 2335 CGTWDAGLRVWVF 1952TGCGGAACATGGGATGCCGGCCTGCGTGTTTGGGTGTTC 2336 CGTWDAGLREIF 1953TGCGGAACATGGGATGCCGGCCTGAGGGAAATTTTC 2337 CGTWASSLSSWVF 1954TGCGGAACATGGGCCAGCAGCCTGAGTTCTTGGGTGTTC 2338 CGTWAGSLSGHVF 1955TGCGGAACATGGGCTGGCAGCCTGAGTGGTCATGTCTTC 2339 CGTWAGSLSAAWVF 1956TGCGGAACATGGGCTGGCAGCCTGAGTGCCGCTTGGGTGTTC 2340 CGTWAGSLNVYWVF 1957TGCGGAACATGGGCTGGCAGCCTGAATGTTTATTGGGTGTTC 2341 CGTWAGNLRPNWVF 1958TGCGGAACATGGGCTGGCAACCTGAGACCTAATTGGGTGTTC 2342 CGTRGSLGGAVF 1959TGCGGAACAAGGGGTAGCCTGGGTGGTGCGGTGTTC 2343 CGTRDTTLSVPVF 1960TGCGGAACAAGGGATACCACCCTGAGTGTCCCGGTGTTC 2344 CGTRDTSLNIEIF 1961TGCGGAACACGGGATACCAGCCTCAATATTGAAATCTTC 2345 CGTRDTSLNDVF 1962TGTGGAACACGGGATACCAGCCTGAATGATGTCTTC 2346 CGTRDTRLSIVVF 1963TGCGGAACACGGGATACCCGCCTGAGTATTGTGGTTTTC 2347 CGTRDTILSAEVF 1964TGCGGCACACGGGATACCATCCTGAGTGCTGAGGTGTTC 2348 CGTRDRSLSGWVF 1965TGCGGAACACGGGATAGAAGCCTGAGTGGTTGGGTGTTC 2349 CGSWYYNVFLF 1966TGCGGATCATGGTATTACAATGTCTTCCTTTTC 2350 CGSWHSSLNLVVF 1967TGCGGATCTTGGCATAGCAGCCTCAACCTTGTCGTCTTC 2351 CGSWGSGLSAPYVF 1968TGCGGATCATGGGGTAGTGGCCTGAGTGCCCCTTATGTCTTC 2352 CGSWESGLGAWLF 1969TGCGGTTCGTGGGAAAGCGGCCTGGGTGCTTGGCTGTTC 2353 CGSWDYGLLLF 1970TGCGGATCCTGGGATTACGGCCTCCTACTCTTC 2354 CGSWDVSLTAVF 1971TGCGGTTCATGGGATGTCAGCCTGACTGCTGTTTTC 2355 CGSWDVSLNVGIF 1972TGCGGATCCTGGGATGTCAGTCTCAATGTTGGCATTTTC 2356 CGSWDTTLRAWVF 1973TGCGGATCATGGGATACCACCCTGCGTGCTTGGGTGTTC 2357 CGSWDTSPVRAWVF 1974TGCGGCTCGTGGGATACCAGCCCTGTCCGTGCTTGGGTGTTC 2358 CGSWDTSLSVWVF 1975TGCGGATCATGGGATACCAGCCTGAGTGTTTGGGTGTTC 2359 CGSWDTSLSAEVF 1976TGCGGATCATGGGATACCAGCCTGAGTGCTGAGGTGTTC 2360 CGSWDTSLRAWVF 1977TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGTGTTC 2361 CGSWDTSLRAWAF 1978TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGCGTTC 2362 CGSWDTSLDARLF 1979TGCGGATCATGGGATACCAGCCTGGATGCTAGGCTGTTC 2363 CGSWDTILLVYVF 1980TGCGGATCATGGGATACCATCCTGCTTGTCTATGTCTTC 2364 CGSWDRWQAAVF 1981TGCGGATCATGGGATCGCTGGCAGGCTGCTGTCTTC 2365 CGSWDRSLSGYVF 1982TGCGGATCATGGGATAGGAGCCTGAGTGGGTATGTCTTC 2366 CGSWDRSLSAYVF 1983TGCGGATCATGGGATAGAAGCCTGAGTGCTTATGTCTTC 2367 CGSWDRSLSAVVF 1984TGCGGATCATGGGATAGGAGCCTGAGTGCCGTGGTTTTC 2368 CGSWDNTLGVVLF 1985TGCGGATCATGGGATAACACCTTGGGTGTTGTTCTCTTC 2369 CGSWDNRLSTVIF 1986TGCGGATCGTGGGATAACAGACTAAGTACTGTCATCTTC 2370 CGSWDNRLNTVIF 1987TGCGGAAGCTGGGATAATCGATTGAACACTGTGATTTTC 2371 CGSWDLSPVRVLVF 1988TGCGGTTCATGGGATCTCAGCCCTGTACGTGTCCTTGTGTTC 2372 CGSWDLSLSAVVF 1989TGCGGATCATGGGATCTCAGCCTGAGTGCTGTCGTTTTC 2373 CGSWDKNLRAVLF 1990TGCGGATCATGGGATAAAAACCTGCGTGCTGTGCTGTTC 2374 CGSWDISLSAGVF 1991TGCGGCTCATGGGATATCAGCCTGAGTGCTGGGGTGTTC 2375 CGSWDIRLSAEVF 1992TGCGGATCATGGGATATCAGACTGAGTGCAGAGGTCTTC 2376 CGSWDIKLNIGVF 1993TGCGGATCATGGGACATCAAACTGAATATTGGGGTATTC 2377 CGSWDFSLNYFVF 1994TGCGGATCATGGGATTTCAGTCTCAATTATTTTGTCTTC 2378 CGSWDASLSTEVF 1995TGCGGATCATGGGATGCCAGCCTGAGTACTGAGGTGTTC 2379 CGSWDAGLRGWVF 1996TGCGGATCCTGGGATGCCGGCCTGCGTGGCTGGGTTTTC 2380 CGRWESSLGAVVF 1997TGCGGAAGATGGGAGAGCAGCCTGGGTGCTGTGGTTTTC 2381 CGRWDFSLSAYVF 1998TGCGGAAGATGGGATTTTAGTCTGAGTGCTTATGTCTTC 2382 CGQWDNDLSVWVF 1999TGCGGACAATGGGATAACGACCTGAGTGTTTGGGTGTTC 2383 CGPWHSSVTSGHVL 2000TGCGGACCCTGGCATAGCAGCGTGACTAGTGGCCACGTGCTC 2384 CGLWDASLSAPTWVF 2001TGCGGATTATGGGATGCCAGCCTGAGTGCTCCTACTTGGGTGTTC 2385 CGIWHTSLSAWVF 2002TGTGGAATATGGCACACTAGCCTGAGTGCTTGGGTGTTC 2386 CGIWDYSLDTWVF 2003TGCGGAATATGGGATTACAGCCTGGATACTTGGGTGTTC 2387 CGIWDTSLSAWVF 2004TGCGGCATATGGGATACCAGCCTGAGTGCTTGGGTGTTC 2388 CGIWDTRLSVYVF 2005TGCGGAATTTGGGATACCAGGCTGAGTGTTTATGTCTTC 2389 CGIWDTRLSVYIF 2006TGCGGAATTTGGGATACCAGGCTGAGTGTTTATATCTTC 2390 CGIWDTNLGYLF 2007TGTGGAATATGGGATACGAATCTGGGTTATCTCTTC 2391 CGIWDTGLSAVVF 2008TGCGGTATATGGGATACCGGCCTGAGTGCTGTGGTATTC 2392 CGIWDRSLSAWVF 2009TGCGGAATATGGGATCGCAGCCTGAGTGCTTGGGTGTTT 2393 CGIRDTRLSVYVF 2010TGCGGAATTCGGGATACCAGGCTGAGTGTTTATGTCTTC 2394 CGGWSSRLGVGPVF 2011TGCGGAGGATGGAGTAGCAGACTGGGTGTTGGCCCAGTGTTT 2395 CGGWGSGLSAWVF 2012TGCGGAGGATGGGGTAGCGGCCTGAGTGCTTGGGTGTTC 2396 CGGWDTSLSAWVF 2013TGCGGAGGATGGGATACCAGCCTGAGTGCTTGGGTGTTC 2397 CGGWDRGLDAWVF 2014TGCGGAGGATGGGATAGGGGCCTGGATGCTTGGGTTTTC 2398 CGAWRNNVWVF 2015TGCGGAGCATGGCGTAATAACGTGTGGGTGTTC 2399 CGAWNRRLNPHSHWVF 2016TGCGGAGCATGGAACAGGCGCCTGAATCCTCATTCTCATTGGGTG TTC 2400 CGAWHNKLSAVF 2017TGCGGAGCCTGGCACAACAAACTGAGCGCGGTCTTC 2401 CGAWGSSLRASVF 2018TGCGGAGCATGGGGTAGCAGCCTGAGAGCTAGTGTCTTC 2402 CGAWGSGLSAWVF 2019TGCGGAGCATGGGGTAGCGGCCTGAGTGCTTGGGTGTTC 2403 CGAWESSLSAPYVF 2020TGCGGAGCATGGGAAAGTAGCCTGAGTGCCCCTTATGTCTTC 2404 CGAWESSLNVGLI 2021TGCGGAGCATGGGAGAGCAGCCTCAATGTTGGACTGATC 2405 CGAWESGRSAGVVF 2022TGCGGAGCATGGGAGAGCGGCCGGAGTGCTGGGGTGGTGTTC 2406 CGAWDYSVSGWVF 2023TGCGGAGCTTGGGATTACAGTGTGAGTGGTTGGGTGTTC 2407 CGAWDYSLTAGVF 2024TGCGGAGCATGGGATTACAGCCTGACTGCCGGAGTATTC 2408 CGAWDYRLSAVLF 2025TGCGGAGCCTGGGATTACAGACTGAGTGCCGTGCTATTC 2409 CGAWDVRLDVGVF 2026TGCGGAGCGTGGGATGTTCGTCTGGATGTTGGGGTGTTC 1203 CGAWDTYSYVF 2027TGCGGAGCATGGGATACCTACAGTTATGTCTTC 1204 CGAWDTTLSGVVF 2028TGCGGAGCATGGGATACGACCCTGAGTGGTGTGGTATTC 1205 CGAWDTTLSAVIF 2029TGCGGAGCGTGGGATACTACCCTGAGTGCTGTGATATTC 1206 CGAWDTSQGASYVF 2030TGCGGCGCATGGGATACCAGCCAGGGTGCGTCTTATGTCTTT 1207 CGAWDTSPVRAGVF 2031TGCGGAGCATGGGATACCAGCCCTGTACGTGCTGGGGTGTTC 1208 CGAWDTSLWLF 2032TGCGGAGCATGGGATACCAGCCTGTGGCTTTTC 1209 CGAWDTSLTVYVF 2033TGCGGAGCATGGGATACCAGCCTGACTGTTTATGTCTTC 1210 CGAWDTSLTAGVF 2034TGCGGAGCATGGGACACCAGTCTGACTGCTGGGGTGTTC 1211 CGAWDTSLSTVVF 2035TGCGGAGCTTGGGATACCAGCCTGAGTACTGTGGTTTTC 1212 CGAWDTSLSSRYIF 2036TGCGGAGCATGGGATACCAGCCTGAGTTCTAGATACATATTC 1213 CGAWDTSLSGYVF 2037TGCGGAGCATGGGATACCAGCCTGAGTGGTTATGTCTTC 1214 CGAWDTSLSGWVF 2038TGCGGAGCCTGGGATACCAGCCTGAGTGGCTGGGTGTTC 1215 CGAWDTSLSGVLF 2039TGCGGAGCATGGGATACCAGTCTGAGTGGTGTGCTATTC 1216 CGAWDTSLSGLVF 2040TGCGGAGCTTGGGATACCAGCTTGAGTGGTCTTGTTTTC 1217 CGAWDTSLSGFVF 2041TGCGGAGCTTGGGATACCAGCTTGAGTGGTTTTGTTTTC 1218 CGAWDTSLSGEVF 2042TGCGGAGCATGGGATACCAGCCTGAGTGGTGAGGTCTTT 1219 CGAWDTSLSDFVF 2043TGCGGAGCTTGGGATACCAGCTTGAGTGATTTTGTTTTC 1220 CGAWDTSLRTAIF 2044TGCGGAGCATGGGATACCAGCCTGCGAACTGCGATATTC 1221 CGAWDTSLRLF 2045TGCGGAGCATGGGATACCAGCCTGCGGCTTTTC 1222 CGAWDTSLNVHVF 2046TGCGGAGCATGGGATACCAGCCTGAATGTTCATGTCTTC 1223 CGAWDTSLNKWVF 2047TGCGGAGCATGGGATACCAGCCTCAATAAATGGGTGTTC 1224 CGAWDTRLSARLF 2048TGCGGAGCATGGGATACCCGCCTCAGTGCGCGGCTGTTC 1225 CGAWDTRLRGF1F 2049TGCGGAGCATGGGATACCAGACTGAGGGGTTTTATTTTC 1226 CGAWDTNLGNVLL 2050TGCGGAGCATGGGATACTAATTTGGGGAATGTTCTCCTC 1227 CGAWDTNLGKWVF 2051TGCGGGGCATGGGATACCAACCTGGGTAAATGGGTTTTC 1228 CGAWDTGLEWYVF 2052TGCGGAGCATGGGATACCGGCCTTGAGTGGTATGTTTTT 1229 CGAWDRTSGLWLF 2053TGCGGAGCATGGGATAGGACTTCTGGATTGTGGCTTTTC 1230 CGAWDRSLVAGLF 2054TGCGGAGCGTGGGATCGTAGCCTGGTTGCTGGACTCTTC 1231 CGAWDRSLTVYVF 2055TGCGGAGCGTGGGATAGAAGCCTGACTGTTTATGTCTTC 1232 CGAWDRSLSGYVF 2056TGCGGAGCATGGGATAGAAGCCTGAGTGGTTATGTCTTC 1233 CGAWDRSLSAYVF 2057TGCGGAGCATGGGATAGAAGCCTGAGTGCTTATGTCTTC 1234 CGAWDRSLSAVVF 2058TGCGGAGCATGGGATAGAAGCCTGAGTGCGGTGGTATTC 1235 CGAWDRSLSAGVF 2059TGCGGAGCATGGGATCGCAGCCTGAGTGCTGGGGTTTTC 1236 CGAWDRSLRIVVF 2060TGCGGAGCGTGGGATCGCAGCCTGCGTATTGTGGTATTC 1237 CGAWDRSLRAYVF 2061TGCGGAGCATGGGATAGAAGTCTGAGGGCTTACGTCTTC 1238 CGAWDRSLNVWLF 2062TGCGGAGCATGGGATAGAAGTCTGAATGTTTGGCTGTTC 1239 CGAWDRGLNVGWLF 2063TGCGGCGCCTGGGATAGGGGCCTGAATGTCGGTTGGCTTTTC 1240 CGAWDNRLSILAF 2064TGCGGCGCATGGGATAATAGACTGAGTATTTTGGCCTTC 1241 CGAWDNDLTAYVF 2065TGCGGAGCTTGGGATAATGACCTGACAGCTTATGTCTTC 1242 CGAWDFSLTPLF 2066TGCGGGGCATGGGATTTCAGCCTGACTCCTCTCTTC 1243 CGAWDDYRGVSIYVF 2067TGCGGAGCCTGGGATGACTATCGGGGTGTGAGTATTTATGTCTTC 1244 CGAWDDRPSSAVVF 2068TGTGGAGCATGGGATGACCGGCCTTCGAGTGCCGTGGTTTTC 1245 CGAWDDRLTVVVF 2069TGCGGAGCATGGGATGACAGACTGACTGTCGTTGTTTTC 1246 CGAWDDRLGAVF 2070TGCGGAGCGTGGGATGACAGGCTGGGTGCTGTGTTC 1247 CGAWDASLNPGRAF 2071TGCGGAGCGTGGGATGCCAGCCTGAATCCTGGCCGGGCATTC 1248 CGAWDAGLREIF 2072TGCGGAGCATGGGATGCCGGCCTGAGGGAAATTTTC 1249 CGAWAGSPSPWVF 2073TGCGGAGCTTGGGCTGGCAGTCCGAGTCCTTGGGTTTTC 1250 CGAFDTTLSAGVF 2074TGCGGAGCATTCGACACCACCCTGAGTGCTGGCGTTTTC 1251 CETWESSLSVGVF 2075TGCGAAACATGGGAGAGCAGCCTGAGTGTTGGGGTCTTC 1252 CETWESSLRVWVF 2076TGCGAAACATGGGAAAGCAGCCTGAGGGTTTGGGTGTTC 1253 CETWDTSLSGGVF 2077TGCGAAACGTGGGATACCAGCCTGAGTGGTGGGGTGTTC 1254 CETWDTSLSDFYVF 2078TGCGAAACATGGGATACCAGCCTGAGTGACTTTTATGTCTTC 1255 CETWDTSLSALF 2079TGCGAAACATGGGATACCAGCCTGAGTGCCCTCTTC 1256 CETWDTSLRAEVF 2080TGCGAAACATGGGATACCAGCCTGCGTGCTGAAGTCTTC 1257 CETWDTSLNVVVF 2081TGCGAAACATGGGATACCAGCCTGAATGTTGTGGTATTC 1258 CETWDTSLGAVVF 2082TGCGAAACATGGGATACCAGCCTGGGTGCCGTGGTGTTC 1259 CETWDRSLSGVVF 2083TGCGAAACATGGGATAGAAGCCTGAGTGGTGTGGTATTC 1260 CETWDRSLSAWVF 2084TGCGAAACATGGGATAGGAGCCTGAGTGCTTGGGTGTTT 1261 CETWDRSLSAVVF 2085TGCGAAACATGGGATCGCAGCCTGAGTGCTGTGGTCTTC 1262 CETWDRGLSVVVF 2086TGCGAGACGTGGGATAGAGGCCTGAGTGTTGTGGTTTTC 1263 CETWDRGLSAVVF 2087TGCGAAACATGGGATAGGGGCCTGAGTGCAGTGGTATTC 1264 CETWDHTLSVVIF 2088TGCGAAACATGGGATCACACCCTGAGTGTTGTGATATTC 1265 CETWDASLTVVLF 2089TGCGAAACATGGGATGCCAGCCTGACTGTTGTGTTATTC 1266 CETWDASLSAGVF 2090TGCGAAACATGGGATGCCAGCCTGAGTGCTGGGGTGTTC 1267 CETWDAGLSEVVF 2091TGCGAAACGTGGGATGCCGGCCTGAGTGAGGTGGTGTTC 1268 CE1FDTSLSVVVF 2092TGCGAAACATTTGATACCAGCCTGAGTGTTGTAGTCTTC 1269 CE1FDTSLNIVVF 2093TGCGAAACATTTGATACCAGCCTAAATATTGTAGTCTTT 1270 CESWDRSRIGVVF 2094TGCGAATCATGGGATAGAAGCCGGATTGGTGTGGTCTTC 1271 CESWDRSLSARVY 2095TGCGAAAGTTGGGACAGGAGTCTGAGTGCCCGGGTGTAC 1272 CESWDRSLRAVVF 2096TGCGAATCCTGGGATAGGAGCCTGCGTGCCGTGGTCTTC 1273 CESWDRSLIVVF 2097TGCGAATCTTGGGATCGTAGTTTGATTGTGGTGTTC 1274 CESWDNNLNEVVF 2098TGCGAAAGTTGGGATAACAATTTAAATGAGGTGGTTTTC 1275 CEIWESSPSADDLVF 2099TGCGAAATATGGGAGAGCAGCCCGAGTGCTGACGATTTGGTGTTC 1276 CEAWDTSLSGAVF 2100TGCGAAGCATGGGATACCAGCCTGAGTGGTGCGGTGTTC 1277 CEAWDTSLSAGVF 2101TGCGAAGCATGGGATACCAGCCTGAGTGCCGGGGTGTTC 1278 CEAWDTSLGGGVF 2102TGCGAAGCATGGGATACCAGCCTGGGTGGTGGGGTGTTC 1279 CEAWDRSLTGSLF 2103TGCGAAGCATGGGATCGCAGCCTGACTGGTAGCCTGTTC 1280 CEAWDRGLSAVVF 2104TGCGAAGCGTGGGATAGGGGCCTGAGTGCAGTGGTATTC 1281 CEAWDNILSTVVF 2105TGCGAAGCCTGGGATAACATCCTGAGTACTGTGGTGTTC 1282 CEAWDISLSAGVF 2106TGCGAAGCATGGGACATCAGCCTGAGTGCTGGGGTGTTC 1283 CEAWDADLSGAVF 2107TGCGAAGCATGGGATGCCGACCTGAGTGGTGCGGTGTTC 1284 CATWTGSFRTGHYVF 2108TGCGCAACATGGACTGGTAGTTTCAGAACTGGCCATTATGTCTTC 1285 CATWSSSPRGWVF 2109TGCGCAACATGGAGTAGCAGTCCCAGGGGGTGGGTGTTC 1286 CATWHYSLSAGRVF 2110TGCGCAACATGGCATTACAGCCTGAGTGCTGGCCGAGTGTTC 1287 CATWHTSLSIVQF 2111TGCGCAACATGGCATACCAGCCTGAGTATTGTGCAGTTC 1288 CATWHSTLSADVLF 2112TGCGCAACATGGCATAGCACCCTGAGTGCTGATGTGCTTTTC 1289 CATWHSSLSAGRLF 2113TGCGCAACATGGCATAGCAGCCTGAGTGCTGGCCGACTCTTC 1290 CATWHIARSAWVF 2114TGCGCAACATGGCATATCGCTCGGAGTGCCTGGGTGTTC 1291 CATWGSSQSAVVF 2115TGCGCAACATGGGGTAGTAGTCAGAGTGCCGTGGTATTC 1292 CATWGSSLSAGGVF 2116TGCGCAACATGGGGTAGCAGCCTGAGTGCTGGGGGTGTTTTC 1293 CATWEYSLSVVLF 2117TGTGCAACATGGGAATACAGCCTGAGTGTTGTGCTGTTC 1294 CATWETTRRASFVF 2118TGCGCAACATGGGAGACCACCCGACGTGCCTCTTTTGTCTTC 1295 CATWETSLNVYVF 2119TGCGCAACATGGGAGACCAGCCTGAATGTTTATGTCTTC 1296 CATWETSLNVVVF 2120TGCGCAACATGGGAAACTAGCCTGAATGTTGTGGTCTTC 1297 CATWETSLNLYVF 2121TGCGCAACATGGGAGACCAGCCTGAATCTTTATGTCTTC 1298 CATWETGLSAGEVF 2122TGCGCAACATGGGAGACTGGCCTAAGTGCTGGAGAGGTGTTC 1299 CATWESTLSVVVF 2123TGCGCGACGTGGGAGAGTACCCTAAGTGTTGTGGTTTTC 1300 CATWESSLSIFVF 2124TGCGCAACGTGGGAGAGCAGCCTGAGTATTTTTGTCTTC 1301 CATWESSLNTFYVF 2125TGCGCAACATGGGAAAGCAGCCTCAACACTTTTTATGTCTTC 1302 CATWESRVDTRGLLF 2126TGCGCAACATGGGAGAGTAGGGTGGATACTCGAGGGTTGTTATTC 1303 CATWESGLSGAGVF 2127TGCGCAACATGGGAGAGCGGCCTGAGTGGTGCGGGGGTGTTC 1304 CATWEGSLNTFYVF 2128TGCGCAACATGGGAAGGCAGCCTCAACACTTTTTATGTCTTC 1305 CATWDYSLSAVVF 2129TGCGCAACTTGGGATTATAGCCTGAGTGCTGTGGTGTTC 1306 CATWDYRLSIVVF 2130TGCGCAACATGGGATTACAGACTGAGTATTGTGGTATTC 1307 CATWDYNLGAAVF 2131TGCGCAACATGGGATTATAACCTGGGAGCTGCGGTGTTC 1308 CATWDVTLGVLHF 2132TGCGCCACATGGGATGTCACCCTGGGTGTCTTGCATTTC 1309 CATWDTTLSVWVF 2133TGCGCAACATGGGATACAACACTGAGTGTCTGGGTCTTC 1310 CATWDTTLSVVLF 2134TGCGCAACATGGGATACCACCCTGAGTGTAGTACTTTTC 1311 CATWDTTLSVEVF 2135TGCGCAACATGGGATACCACCCTGAGTGTTGAGGTCTTC 1312 CATWDTSPSLSGFWVF 2136TGCGCAACATGGGATACCAGCCCCAGCCTGAGTGGTTTTTGGGTG TTC 1313 CATWDTSLTGVVF2137 TGCGCAACATGGGATACCAGCCTGACTGGTGTGGTATTC 1314 CATWDTSLTGAVF 2138TGCGCAACATGGGATACCAGCCTGACTGGTGCGGTGTTC 1315 CATWDTSLTAWVF 2139TGCGCAACATGGGATACCAGCCTGACTGCCTGGGTATTC 1316 CATWDTSLTAVVF 2140TGCGCAACATGGGATACCAGCCTGACTGCTGTGGTTTTC 1317 CATWDTSLTAKVF 2141TGCGCAACATGGGATACTAGCCTGACTGCTAAGGTGTTC 1318 CATWDTSLSVVVF 2142TGCGCAACATGGGACACCAGCCTGAGTGTTGTGGTTTTC 1319 CATWDTSLSVGVF 2143TGCGCTACTTGGGATACCAGCCTGAGTGTTGGGGTATTT 1320 CATWDTSLSSWVF 2144TGCGCAACATGGGATACCAGCCTGAGTTCTTGGGTGTTC 1321 CATWDTSLSGGVL 2145TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTACTC 1322 CATWDTSLSGGVF 2146TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTGTTC 1323 CATWDTSLSGGRVF 2147TGCGCAACATGGGATACCAGCCTGAGTGGTGGCCGAGTGTTC 1324 CATWDTSLSGDRVF 2148TGCGCAACATGGGATACCAGCCTGAGTGGTGACCGAGTGTTC 1325 CATWDTSLSEGVF 2149TGCGCAACGTGGGATACTAGCCTGAGTGAAGGGGTGTTC 1326 CATWDTSLSAVVL 2150TGCGCAACCTGGGATACCAGCCTGAGTGCCGTGGTGCTC 1327 CATWDTSLSAVF 2151TGCGCAACATGGGATACCAGCCTGAGTGCTGTCTTC 1328 CATWDTSLSARVF 2152TGCGCGACATGGGATACCAGCCTGAGTGCTCGGGTGTTC 1329 CATWDTSLSALF 2153TGCGCAACATGGGATACCAGCCTGAGTGCCTTATTC 1330 CATWDTSLSAHVF 2154TGCGCAACATGGGATACCAGCCTGAGTGCTCATGTCTTC 1331 CATWDTSLSAGRVF 2155TGCGCAACATGGGATACCAGCCTGAGTGCTGGCCGGGTGTTC 1332 CATWDTSLSAEVF 2156TGCGCAACATGGGATACCAGCCTGAGTGCGGAGGTCTTC 1333 CATWDTSLSADAGGGV 2157TGCGCAACATGGGATACCAGCCTGAGTGCTGATGCTGGTGGGGGG F GTCTTC 1334CATWDTSLRVVVF 2158 TGCGCAACATGGGATACCAGCCTGCGTGTCGTGGTATTC 1335CATWDTSLRGVF 2159 TGCGCAACATGGGATACCAGCCTGAGAGGGGTGTTC 1336CATWDTSLPAWVF 2160 TGCGCAACATGGGATACCAGCCTGCCTGCGTGGGTGTTC 1337CATWDTSLNVGVF 2161 TGTGCAACATGGGATACCAGCCTGAATGTTGGGGTATTC 1338CATWDTSLGIVLF 2162 TGCGCAACATGGGATACCAGCCTGGGTATTGTGTTATTT 1339CATWDTSLGARVVF 2163 TGCGCAACATGGGACACCAGCCTGGGTGCGCGTGTGGTCTTC 1340CATWDTSLGALF 2164 TGTGCAACGTGGGATACCAGTCTAGGTGCCTTGTTC 1341CATWDTSLATGLF 2165 TGCGCAACATGGGATACCAGCCTGGCGACTGGACTGTTC 1342CATWDTSLAAWVF 2166 TGCGCAACATGGGATACCAGCCTGGCTGCCTGGGTATTC 1343CATWDTRLSAVVF 2167 TGCGCAACCTGGGATACCAGGCTGAGTGCTGTGGTCTTC 1344CATWDTRLSAGVF 2168 TGCGCAACATGGGATACCAGGCTGAGTGCTGGGGTGTTC 1345CATWDTRLLITVF 2169 TGTGCAACGTGGGACACACGTCTACTTATTACGGITTTC 1346CATWDTLLSVELF 2170 TGCGCAACATGGGACACCCTCCTGAGTGTTGAACTCTTC 1347CATWDTGRNPHVVF 2171 TGCGCAACATGGGATACTGGCCGCAATCCTCATGTGGTCTTC 1348CATWDTGLSSVLF 2172 TGCGCAACATGGGATACCGGCCTGTCTTCGGTGTTGTTC 1349CATWDTGLSAVF 2173 TGCGCAACGTGGGATACCGGCCTGAGTGCGGTTTTC 1350CATWDRTLSIGVF 2174 TGCGCTACGTGGGATAGGACCCTGAGTATTGGAGTCTTC 1351CATWDRSVTAVLF 2175 TGCGCAACGTGGGATCGCAGTGTGACTGCTGTGCTCTTC 1352CATWDRSLSGVVF 2176 TGCGCAACCTGGGATAGGAGCCTGAGTGGTGTGGTGTTC 1353CATWDRSLSAVVF 2177 TGCGCAACATGGGATAGAAGCCTGAGTGCTGTGGTCTTC 1354CATWDRSLSAVPWVF 2178 TGCGCAACATGGGATAGAAGCCTGAGTGCTGTTCCTTGGGTGTTC 1355CATWDRSLSAGVF 2179 TGCGCAACATGGGATCGCAGCCTGAGTGCTGGGGTGTTC 1356CATWDRSLRAGVF 2180 TGCGCAACGTGGGATAGGAGCCTGCGTGCTGGGGTGTTC 1357CATWDRSLNVYVL 2181 TGCGCAACATGGGATCGCAGTCTGAATGTTTATGTCCTC 1358CATWDRILSAEVF 2182 TGCGCAACGTGGGATCGCATCCTGAGCGCTGAGGTGTTC 1359CATWDRGLSTGVF 2183 TGCGCAACGTGGGATAGAGGCCTGAGTACTGGGGTGTTC 1360CATWDNYLGAAVF 2184 TGCGCAACATGGGATAACTACCTGGGTGCTGCCGTGTTC 1361CATWDNTPSNIVVF 2185 TGCGCAACATGGGATAACACGCCTTCGAATATTGTGGTATTC 1362CATWDNTLSVWVF 2186 TGCGCAACATGGGATAATACACTGAGTGTGTGGGTCTTC 1363CATWDNTLSVNWVF 2187 TGCGCAACATGGGATAACACCCTGAGTGTCAATTGGGTGTTC 1364CATWDNTLNVFYVF 2188 TGCGCAACCTGGGATAACACACTGAATGTCTTTTATGTTTTC 1365CATWDNRLSSVVF 2189 TGTGCGACATGGGATAATCGGCTCAGTTCTGTGGTCTTC 1366CATWDNRLSAGVL 2190 TGCGCAACATGGGATAACCGCCTGAGTGCTGGGGTGCTC 1367CATWDNRLSAGVF 2191 TGCGCAACGTGGGATAACAGGCTGAGTGCTGGGGTGTTC 1368CATWDNRDWVF 2192 TGCGCAACATGGGATAACAGGGATTGGGTCTTC 1369 CATWDNNLGAGVF2193 TGCGCAACATGGGATAACAACCTGGGTGCTGGGGTGTTC 1370 CATWDNKLTSGVF 2194TGCGCAACATGGGATAACAAGCTGACTTCTGGGGTCTTC 1371 CATWDNILSAWVF 2195TGCGCAACATGGGATAACATCCTGAGTGCCTGGGTGTTT 1372 CATWDNDIHSGLF 2196TGCGCAACCTGGGACAACGATATACATTCTGGGCTGTTC 1373 CATWDLSLSALF 2197TGCGCAACTTGGGATCTCAGCCTGAGTGCCCTGTTC 1374 CATWDITLSAEVF 2198TGCGCAACATGGGATATCACCCTGAGTGCTGAGGTGTTC 1375 CATWDISPSAGGVF 2199TGCGCAACGTGGGATATCAGCCCGAGTGCTGGCGGGGTGTTC 1376 CATWDISLSTGRAVF 2200TGCGCAACATGGGATATCAGTCTAAGTACTGGCCGGGCTGTGTTC 1377 CATWDISLSQVF 2201TGCGCAACATGGGATATCAGTCTGAGTCAGGTATTC 1378 CATWDIRLSSGVF 2202TGCGCAACATGGGATATCAGGCTGAGTAGTGGAGTGTTC 1379 CATWDIGPSAGGVF 2203TGCGCAACGTGGGATATCGGCCCGAGTGCTGGCGGGGTGTTC 1380 CATWDHSRAGVLF 2204TGCGCAACATGGGATCACAGCCGGGCTGGTGTGCTATTC 1381 CATWDHSPSVGEVF 2205TGCGCAACATGGGATCACAGTCCGAGTGTTGGAGAAGTCTTC 1382 CATWDHSLRVGVF 2206TGCGCAACATGGGATCACAGCCTGCGTGTTGGGGTGTTC 1383 CATWDHSLNIGVF 2207TGCGCAACATGGGATCACAGCCTGAACATTGGGGTGTTC 1384 CATWDHSLGLWAF 2208TGCGCAACATGGGATCACAGCCTGGGTCTTTGGGCATTC 1385 CATWDHNLRLVF 2209TGCGCCACATGGGATCACAATCTGCGTCTTGTTTTC 1386 CATWDHILASGVF 2210TGCGCGACTTGGGATCACATCCTGGCTTCTGGGGTGTTC 1387 CATWDFSLSVWVF 2211TGCGCAACATGGGATTTCAGCCTGAGTGTTTGGGTGTTC 1388 CATWDFSLSAWVF 2212TGCGCAACATGGGATTTCAGCCTGAGTGCTTGGGTGTTC 1389 CATWDDTLTAGVF 2213TGCGCAACATGGGATGACACCCTCACTGCTGGTGTGTTC 1390 CATWDDRLSAVLF 2214TGCGCAACATGGGACGACAGGCTGAGTGCTGTGCTTTTC 1391 CATWDDRLDAAVF 2215TGCGCAACATGGGATGACAGGCTGGATGCTGCGGTGTTC 1392 CATWDATLNTGVF 2216TGCGCAACATGGGATGCGACCCTGAATACTGGGGTGTTC 1393 CATWDASLSVWLL 2217TGCGCAACATGGGATGCCAGCCTGAGTGTTTGGCTGCTC 1394 CATWDASLSGGVF 2218TGCGCGACATGGGATGCCAGCCTGAGTGGTGGGGTGTTC 1395 CATRDTTLSAVLF 2219TGCGCAACACGGGATACCACCCTCAGCGCCGTTCTGTTC 1396 CATLGSSLSLWVF 2220TGCGCTACATTGGGTAGTAGCCTGAGTCTCTGGGTGTTC 1397 CATIETSLPAWVF 2221TGCGCAACAATCGAAACTAGCCTGCCTGCCTGGGTATTC 1398 CATGDRSLTVEVF 2222TGCGCAACAGGGGACAGAAGCCTGACTGTTGAGGTATTC 1399 CATGDLGLTIVF 2223TGCGCTACAGGGGATCTCGGCCTGACCATAGTCTTC 1400 CASWDYRGRSGWVF 2224TGCGCATCATGGGATTACAGGGGGAGATCTGGTTGGGTGTTC 1401 CASWDTTLNVGVF 2225TGCGCATCATGGGATACCACCCTGAATGTTGGGGTGTTC 1402 CASWDTTLGFVLF 2226TGCGCTTCATGGGATACCACCCTGGGTTTTGTGTTATTC 1403 CASWDTSLSGGYVF 2227TGCGCATCATGGGATACCAGCCTGAGTGGTGGTTATGTCTTC 1404 CASWDTSLRAGVF 2228TGCGCATCATGGGATACCAGCCTCCGTGCTGGGGTGTTC 1405 CASWDTSLGAGVF 2229TGCGCATCATGGGATACCAGCCTGGGTGCTGGGGTGTTC 1406 CASWDRGLSAVVF 2230TGCGCATCATGGGACAGAGGCCTGAGTGCAGTGGTGTTC 1407 CASWDNVLRGVVF 2231TGTGCTAGTTGGGATAACGTCCTGCGTGGTGTGGTATTC 1408 CASWDNRLTAVVF 2232TGCGCGTCATGGGATAACAGGCTGACTGCCGTGGTTTTC 1409 CASWDASLSVAF 2233TGCGCATCATGGGATGCAAGCCTGTCCGTCGCTTTC 1410 CASWDAGLSSYVF 2234TGCGCTTCGTGGGATGCCGGCCTGAGTTCTTATGTCTTC 1411 CASGDTSLSGVIF 2235TGCGCATCCGGGGATACCAGCCTGAGTGGTGTGATATTC 1412 CARWHTSLSIWVF 2236TGCGCAAGATGGCATACGAGCCTAAGTATTTGGGTCTTC 1413 CAIWDTGLSPGQVAF 2237TGCGCAATATGGGATACCGGCCTGAGTCCTGGCCAAGTTGCCTTC 1414 CAAWHSGLGLPVF 2238TGCGCAGCATGGCATAGCGGCCTGGGTCTCCCGGTCTTC 1415 CAAWDYSLSAGVF 2239TGCGCAGCATGGGATTACAGCCTGAGTGCTGGGGTGTTC 1416 CAAWDTTLRVRLF 2240TGCGCAGCCTGGGATACTACCCTGCGTGTTAGGCTGTTC 1417 CAAWDTSLTAWVF 2241TGCGCAGCATGGGATACCAGCCTGACTGCCTGGGTTTTC 1418 CAAWDTSLSGGVF 2242TGCGCAGCATGGGATACCAGCTTGAGTGGTGGGGTGTTC 1419 CAAWDTSLSGEAVF 2243TGCGCAGCATGGGATACCAGCCTGAGTGGCGAGGCTGTGTTC 1420 CAAWDTSLSGAVF 2244TGCGCAGCATGGGATACCAGCTTGAGTGGTGCGGTGTTC 1421 CAAWDTSLSAWVF 2245TGCGCAGCATGGGATACCAGCCTGAGTGCCTGGGTGTTC 1422 CAAWDTSLSAGVF 2246TGCGCAGCATGGGATACCAGCCTGAGTGCTGGGGTATTC 1423 CAAWDTSLDTYVF 2247TGCGCAGCATGGGATACCAGCCTGGATACTTATGTCTTC 1424 CAAWDTRLSGVLF 2248TGCGCTGCATGGGATACCCGTCTGAGTGGTGTGTTATTC 1425 CAAWDTRLSAGVF 2249TGCGCAGCATGGGATACCAGGCTGAGTGCTGGGGTGTTC 1426 CAAWDRSLSTGVF 2250TGCGCAGCATGGGATCGCAGTCTGAGTACTGGAGTTTTC 1427 CAAWDIRRSVLF 2251TGCGCAGCGTGGGATATCCGCCGGTCTGTCCTTTTC 1428 CAAWDHTQRLSF 2252TGCGCTGCGTGGGATCACACTCAGCGTCTTTCCTTC 1429 CAAWDHSLSAGQVF 2253TGCGCAGCATGGGATCACAGCCTGAGTGCTGGCCAGGTGTTC 1430 CAAVDTGLKEWVF 2254TGCGCAGCAGTCGATACTGGTCTGAAAGAATGGGTGTTC

The CDRs were prescreened to contain no amino acid liabilities, crypticsplice sites or nucleotide restriction sites. The CDR variation wasobserved in at least two individuals and comprises the near-germlinespace of single, double and triple mutations. The order of assembly isseen in FIG. 8C.

The VH domains that were designed include IGHV1-69 and IGHV3-30. Each oftwo heavy chain VH domains are assembled with their respective invariant4 framework elements (FW1, FW2, FW3, FW4) and variable 3 CDR (H1, H2,H3) elements. For IGHV1-69, 417 variants were designed for H1 and 258variants were designed for H2. For IGHV3-30, 535 variants were designedfor H1 and 165 variants were designed for H2. For the CDR H3, the samecassette was used in both IGHV1-69 and IGHV-30 since both designed usean identical FW4, and because the edge of FW3 is also identical for bothIGHV1-69 and IGHV3-30. The CDR H3 comprises an N-terminus and C-terminuselement that are combinatorially joined to a central middle element togenerate 1×10¹⁰ diversity. The N-terminal and middle element overlapwith a “GGG” glycine codon. The middle and C-terminal element overlapwith a “GGT” glycine codon. The CDR H3 comprises 5 subpools that wereassembled separately. The various N-terminus and C-terminus elementscomprise sequences as seen in Table 10.

TABLE 10 Sequences for N-terminus and C-terminus elements ElementSEQ ID NO Sequence Stem A 2255 CARDLRELECEEWT XXX SRGPCVDPRGVAGSFDVWStem B 2256 CARDMYYDF XXX EVVPADDAFDIW Stem C 2257CARDGRGSLPRPKGGP XXX YDSSEDSGGAFDIW Stem D 2258 CARANQHF XXX GYHYYGMDVWStem E 2259 CAKHMSMQ XXX RADLVGDAFDVW

Example 9. Enrichment for GPCR GLP1R Binding Proteins

Antibodies having CDR-H3 regions with a variant fragments of GPCRbinding protein that were generated by methods described herein werepanned using cell-based methods to identify variants which are enrichedfor binding to particular GPCRs, as described in Example 4.

Variants of the GLP C-terminus peptide were identified (listed in Table11) that when embedded in the CDR-H3 region of an antibody, wererepeatedly and selectively enriched for binding to GPCR GLP1R.

TABLE 11 Sequences of GLP1 embedded in CDR-H3 SEQ ID NO Sequence 2260CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2261CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2262CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2263CAKHMSMQEGAVTGEGQDAKEFIAWLVKGRVRADLVGDAFDVW 2264WAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2265CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2266CARANQHFYEQEGTFTSDVSSYLEGQAAKEFIAWLVKGGIRGYHYYGMDVW 2267CARANQHFTELHGEGQAAKEFIAWLVKGRGQIDIGYHYYGMDVW 2268CARANQHFLGAGVSSYLEGQAAKEFIAWLVKGDTTGYHYYGMDVW 2269CARANQHFLDKGTFTSDVSSYLEGQAAKEFIAWLVKGIYPGYHYYGMDVW 2270CARANQHFGTLSAGEGQAAKEFIAWLVKGGSQYDSSEDSGGAFDIW 2271CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW 2272CARANQHFGGKGEGQAAKEFIAWLVKGGGSGAGYHYYGMDVW 2273CAKQMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2274CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2275CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2276CAKHMSMQDYLVIGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

Example 10. Analysis of GLP1R Binding Protein Variants

Antibodies having CDR-H3 regions with variant fragments of GLP1R bindingprotein were generated by methods described herein were panned usingcell-based methods to identify variants which are enriched for bindingto GLP1R, as described in Example 4.

Next generation sequence (NGS) enrichment for variants of the GLP1Rpeptides was performed (data not shown). Briefly, phage populations weredeep-sequenced after each round of selection are deep-sequenced tofollow enrichment and identify cross-sample clones. Target specificclones were selected after filtering out CHO background clones from theNGS data. For GLP1R peptides, about 2000 VH and VL pairs were barcodeddirectly from a bacterial colony and sequenced to identify non-identicalclones.

GLP1R-1 variant was analyzed for V gene distribution, J genedistribution, V gene family, and CDR3 counts per length. Frequency of Vgenes IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV3-53, IGHV3-NL1, IGHV3-d,IGHV1-46, IGHV3-h, IGHV1, IGHV3-38, IGHV3-48, IGHV1-18, IGHV1-3, andIGHV3-15 was determined (data not shown). High frequency of IGHV1-69 andIGHV3-30 were observed. Frequency of J genes IGHJ3, IGHJ6, IGHJ, IGHJ4,IGHJ5, mIGHJ, IGHJ2, and IGH1 was also determined (data not shown). Highfrequency of IGHJ3 and IGHJ6 were observed with less frequency of IGHJand IGHJ4 observed. Frequency of V genes IGHV1-69, IGHV3-30, IGHV3-23,IGHV3, IGHV1-46, IGHV3-7, IGHV1, and IGHV1-8 was determined (data notshown). High frequency of IGHV1-69 and IGHV3-30 was observed. Frequencyof J genes IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, and IGH1 wasdetermined (data not shown). High frequency of IGHJ3 and IGHJ6 wasobserved with less frequency of IGHJ and IGHJ4 observed.

H accumulation and frequency were determined for GLP1R-1, GLP1R-2,GLP1R-3, GLP1R-4, and GLP1R-5 (data not shown).

Sequence analytics were performed for GLP1R-1, GLP1R-2, GLP1R-3,GLP1R-4, and GLP1R-5 variants (data not shown).

Cell binding was determined for the GLP1R variants. FIGS. 9A-90 show thecell binding data for GLP1R-2 (FIG. 9A), GLP1R-3 (FIG. 9B), GLP1R-8(FIG. 9C), GLP1R-26 (FIG. 9D), GLP1R-30 (FIG. 9E), GLP1R-56 (FIG. 9F),GLP1R-58 (FIG. 9G), GLP1R-10 (FIG. 9H), GLP1R-25 (FIG. 9I), GLP1R-60(FIG. 9J), GLP1R-70 (FIG. 9K), GLP1R-72 (FIG. 9L), GLP1R-83 (FIG. 9M),GLP1R-93 (FIG. 9N), and GLP1R-98 (FIG. 9O).

GLP1R-3, GLP1R-8, GLP1R-26, GLP1R-56, GLP1R-58 and GLP1R-10 were thenanalyzed for allosteric effects on GLP1-7-36 peptide in a cAMP assay.FIGS. 10A-100 show graphs of the GLP1R variants on inhibition ofGLP1-7-36 peptide induced cAMP activity. GLP1R-3 (FIG. 10B), GLP1R-8(FIG. 10C), GLP1R-26 (FIG. 10D), GLP1R-30 (FIG. 10E), GLP1R-56 (FIG.10F), GLP1R-58 (FIG. 10G), GLP1R-10 (FIG. 10H, right graph), GLP1R-25(FIG. 10I), and GLP1R-60 (FIG. 10J) show allosteric inhibition ofGLP1-7-36 peptide induced cAMP activity. FIG. 10H further shows effectsof GLPR-10 on cAMP signal as compared to exendin-4 (FIG. 10H, leftgraph).

GLP1R variants were tested in a cAMP assay to determine if the variantswere antagonists in blocking exendin-4 induced cAMP activity. FIGS.11A-11G depict cell functional data for GLP1R-2 (FIG. 11A), GLP1R-3(FIG. 11B), GLP1R-8 (FIG. 11C), GLP1R-26 (FIG. 11D), GLP1R-30 (FIG.11E), GLP1R-56 (FIG. 11F), and GLP1R-58 (FIG. 11G).

GLP1R-2, GLP1R-3, GLP1R-8, GLP1R-26, GLP1R-30, GLP1R-56, and GLP1R-58were then analyzed for allosteric effects on exendin-4 in a cAMP assay.FIGS. 12A-12G depict graphs of GLP1R-2 (FIG. 12A), GLP1R-3 (FIG. 12B),GLP1R-8 (FIG. 12C), GLP1R-26 (FIG. 12D), GLP1R-30 (FIG. 12E), GLP1R-56(FIG. 12F), and GLP1R-58 (FIG. 12G) variants on inhibition of Exendin-4peptide induced cAMP activity. Table 12 shows the EC50 (nM) data forExendin-4 alone or with GLP1R-2, GLP1R-3, GLP1R-8, GLP1R-26, GLP1R-30,GLP1R-56, and GLP1R-58.

TABLE 12 EC50 (nM) Data EC50 fold-diff Fxendin-4 alone 0.12 +GLP1R-20.12 1.0 +GLP1R-3 0.63 5.4 +GLP1R-8 0.47 4.0 +GLP1R-26 0.77 6.5+GLP1R-30 0.11 1.0 +GLP1R-56 0.82 7.0 +GLP1R-58 0.27 2.3

FACS screening was performed on GLP1R variants. GLP1R-2, GLP1R-3,GLP1R-8, GLP1R-10, GLP1R-25, GLP1R-26, GLP1R-30, GLP1R-56, GLP1R-58,GLP1R-60, GLP1R-70, GLP1R-72, GLP1R-83, GLP1R-93, and GLP1R-98 wereidentified as seen in Table 13. GLP1R-3, GLP1R-8, GLP1R-56, GLP1R-58,GLP1R-60, GLP1R-72, and GLP1R-83 comprise the GLP1 motif. See FIG. 13.GLP1R-25, GLP1R-30, GLP1R-70, GLP1R-93, and GLP1R-98 comprise the GLP2motif. See FIG. 13. GLP1R-50 and GLP1R-71 comprise the CC chemokine 28motif.

TABLE 13 GLP1R Variants SEQ ID NO Variant Sequence 2277 GLP1R-1CARANQHFVDLYGWHGVPKGYHYYGMDVW 2278 GLP1R-2CARDMYYDFETVVEGIQWYEALKAGKLGEVVPADDAFDIW 2279 GLP1R-3CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2280 GLP1R-8CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2281 GLP1R-10CARANQHFFVPGSLKVWLKGVAPESSSEYDSSEDSGGAFDIW 2282 GLP1R-25CARANQHFLSHAGAARDFINWLIQTKITGLGSGYHYYGMDVW 2283 GLP1R-26CAKHMSMQEGVLQGQIPSTIDWEGLLHLIRADLVGDAFDVW 2284 GLP1R-30CARDMYYDFLKIGDNLAARDFINWLIQTKITDGTDTEVVPADDAFDIW 2285 GLP1R-50CARDGRGSLPRPKGGPKFVPGKHETYGHKTGYRLRPGYHYYGMDVW 2286 GLP1R-56CARANQHFFSGAEGEGQAAKEFIAWLVKGIIPGYHYYGMDVW 2287 GLP1R-58CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW 2288 GLP1R-60CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2289 GLP1R-70CARDGRGSLPRPKGGPPSSGRDFINWLIQTKITDGFRYDSSEDSGGAFDIW 2290 GLP1R-71CARDLRELECEEWTRHGGKKHHGKRQSNRAHQGKHETYGHKTGSLVPSRGPCVDPR GVAGSFDVW 2291GLP1R-72 CARDMYYDFHPEGTFTSDVSSYLEGQAAKEFIAWLVKGSLIYEVVPADDAFDIW 2292GLP1R-80 CARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGTEHGYHYY GMDVW2293 GLP1R-83 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2294 GLP1R-93CARANQHFLSHAGAARDFINWLIQTKITGLGSGYHYYGMDVW 2295 GLP1R-98CARDGRGSLPRPKGGPHSGRLGSGYKSYDSSEDSGGAFDIW *bold corresponds to GLP1 orGLP2 motif

The GLP1R variants were assed for aggregation. Size exclusionchromatography (SEC) was performed on GLP1R-30 and GLP1R-56 variants.82.64% of GLP1R-30 was monomeric (˜150 Kd). 97.4% of GLP1R-56 wasmonomeric (˜150 Kd).

Example 11. GPCR Binding Protein Functionality

For a GPCR binding protein, the top 100-200 scFvs from phage-selectionswere converted to full-length immunoglobulins. After immunoglobulinconversion, the clones were transiently transfected in ExpiCHO toproduce immunoglobulins. Kingfisher and Hamilton were used for batch IgGpurifications followed by lab-chip to collect purity data for allpurified immunoglobulins. High yields and purities were obtained from 10mL cultures as seen in Table 14.

TABLE 14 Immunoglobulin Purity Percentage IgG % Name Purity mAb1 100mAb2 100 mAb3 100 mAb4 100 mAb5 98 mAb6 100 mAb7 97 mAb8 100 mAb9 100mAb10 100 mAb11 100 mAb12 100 mAb13 100 mAb14 100 mAb15 100

Stable cell lines expressing GPCR targets were then generated andconfirmed by FACS (data not shown). Cells expressing >80% of the targetwere then directly used for cell-based selections. Five rounds ofselections were carried out against cells overexpressing target ofinterest. 10⁸ cells were used for each round of selection. Beforeselection on target expressing cells, phage from each round was firstdepleted on 10⁸ CHO background cells. Stringency of selections wasincreased by increasing the number of washes in subsequent rounds ofselection. Enrichment ratios were monitored for each round of selection.

Purified IgGs were tested for cell-binding affinity using FACS (FIGS.14A-14C) and cAMP activity (FIG. 14D). Allosteric inhibition wasobserved.

Purified IgGs were tested using BVP ELISA. BVP ELISA showed some clonescomprising BVP scores comparable to comparator antibodies (data notshown).

Example 12. GLP1R scFv Modulators

This example illustrates identification of GLP1R modulators.

Library Panning

The GPCR1.0/2.0 scFv-phage library was incubated with CHO cells for 1hour at room temperature (RT) to deplete CHO cell binders. Afterincubation, the cells were pelleted by centrifuging at 1,200 rpm for 10minutes to remove the non-specific CHO cell binders. The phagesupernatant, which has been depleted of CHO cell binders, was thentransferred to GLP1R expressing CHO cells. The phage supernatant andGLP1R expressing CHO cells were incubated for 1 hour at RT to select forGLP1R binders. After incubation, the non-binding clones were washed awayby washing with PBS several times. Finally, to selectively elute theagonist clones, the phage bound to the GLP1R cells were competed offwith 1 μM of the natural ligand of GLP1R, GLP1 peptide (residues 7 to36). The clones that eluted off the cells were likely binding to theGLP1 ligand binding epitope on GLP1R. Cells were pelleted bycentrifuging at 1,200 rpm for 10 minutes to remove clones that werestill binding to GLP1R on the cells, and were not binding to theendogenous GLP1 ligand binding site (orthosteric site). The supernatantwas amplified in TG1 E. coli cells for the next round of selection. Thisselection strategy was repeated for five rounds. Amplified phage from around was used as the input phage for the subsequent round, and thestringency of washes were increased in each subsequent round ofselections. After five rounds of selection, 500 clones from each ofround 4 and round 5 were Sanger sequenced to identify clones of GLP1Rmodulators. Seven unique clones were reformatted to IgG2, purified andtested in binding by FACS and functional assays.

Binding Assays

Seven GLP1R scFv clones (GLP1R-238, GLP1R-239, GLP1R-240, GLP1R-241,GLP1R-242, GLP1R-243, and GLP1R-244) and two GLP1R IgGs (pGPCR-GLP1R-43and pGPCR-GLP1R-44, Janssen Biotech, J&J) used as controls were testedin binding assays coupled to flow cytometry analysis. CDR3 sequences(Table 15), heavy chain sequences (Table 16), and light chain sequences(Table 17) for GLP1R-238, GLP1R-239, GLP1R-240, GLP1R-241, GLP1R-242,GLP1R-243, and GLP1R-244 are seen below.

TABLE 15 CDR3 sequences SEQ ID NO. Variant CDR-H3 Sequence 2296GLP1R-238 CARANQHFSQAGRAARVPGPSSSLGPRGYHYYGMDVW 2297 GLP1R-239CAKHMSMQSQGLDNLAARDFINWLIQTKITDGFELSRADLVGDAFDVW 2298 GLP1R-240CARDMYYDFFGLGTFTSDVSSYLEGQAAKEFIAWLVKGVSPEVVPADDAFDIW 2299 GLP1R-241CAKHMSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVW 2300 GLP1R-242CAKHMSMQADTGTFTSDVSSYLEGQAAKEFIAWLVKGEFSSRADLVGDAFDVW 2301 GLP1R-243CARANQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVW 2302 GLP1R-244CARANQHFAATGAGEGQAAKEFIAWLVKGRVEIGYHYYGMDVW *bold correspond to GLP-1 orGLP-2 motif

TABLE 16 Variable Heavy Chain Sequences SEQ ID NO. VariantVariable Heavy Chain Sequence 2303 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGSFSSHAISWVRQA 238PGQGLEWMGGIIPIFGAPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFSQAGRAARVPGPSSSLGPRGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2304 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVESGGGVVQPGRSLRLSCAASGFDFSNYGMHWVRQ 239APGKGLEWVADISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQSQGLDNLAARDFINWLIQTKITDGFELSRADLVGDAFDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2305 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFNNYGISWVRQ 240APGQGLEWMGGIIPVFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFFGLGTFTSDVSSYLEGQAAKEFIAWLVKGVSPEVVPADDAFDIWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2306 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQ 241APGQGLEWMGGIIPIFGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHMSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2307 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYEISWVRQA 242PGQGLEWMGGIIPILGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHMSMQADTGTFTSDVSSYLEGQAAKEFIAWLVKGEFSSRADLVGDAFDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2308 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGINWVRQ 243APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2309 GLP1R-MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 244PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFAATGAGEGQAAKEFIAWLVKGRVEIGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

TABLE 17 Variable Light Chain Sequences SEQ ID NO. VariantVariable Light Chain Sequence 2310 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSTSNIANNYVSWYQQL 238PGTAPKLLIYANNRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGAWDVRLDVGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS 2311 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSTSNIEKNYVSWYQQL 239PGTAPKLLIYGNDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWENRLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2312 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSSIGNNYVSWYQQL 240PGTAPKLLIYANNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWSSSPRGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2313 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGISSNIGNNYVSWYQQL 241PGTAPKLLIYDDDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNILSAAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2314 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSNIENNDVSWYQQL 242PGTAPKLLIYGNDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNTLSAGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2315 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSRSNIGKNYVSWYQQ 243LPGTAPKLLIYENNERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCSSYTTSNTQVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2316 GLP1R-MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNVVSWYQQL 244PGTAPKLLIYDNDKRRSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGSWDTSLSVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Briefly, flag-GLP1R-GFP expressing CHO cells (CHO-GLP1R) and CHO-parentcells were incubated with 100 nM IgG for 1 hour on ice, washed threetimes and incubated with Alexa 647 conjugated goat-anti-human antibody(1:200) for 30 minutes on ice, followed by three washes. All incubationsand washes were performed in buffer containing PBS and 0.5% BSA. Fortitrations, IgG was serially diluted 1:3 starting from 100 nM. Cellswere analyzed by flow cytometry and hits in which IgG was found to bindto CHO-GLP1R were identified by measuring the GFP signal against theAlexa 647 signal. GLP1R-238, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243,and GLP1R-244 were found to bind to CHO-GLP1R. GLP1R-238 bound equallyto CHO-GLP1R and CHO-parent cells and thus appears to be a non-specificbinder. Analyses of binding assays with IgG titrations presented asbinding curves plotting IgG concentrations against MFI (meanfluorescence intensity) are seen in FIGS. 15A-15H. Flow cytometry dataof binding assays presented as dot plots with 100 nM IgG are seen inFIGS. 16A-16I.

Functional Assays

All GLP1R scFv clones, as well as pGPCR-GLP1R-43 and pGPCR-GLP1R-44,were assayed for their potential effects on GLP1R signaling byperforming cAMP assays (Eurofins DiscoverX Corporation). These assaysinvolve CHO cells that were engineered to overexpress naturallyGas-coupled wildtype GLP1R and were designed to detect changes inintracellular cAMP levels in response to agonist stimulation of thereceptor. The technology involved in detecting cAMP levels was a no washgain-of-signal competitive immunoassay based on Enzyme FragmentComplementation technology and produced a luminescent signal that wasdirectly proportional to the amount of cAMP in the cells. Experimentswere designed to determine agonist or allosteric activity of the IgGs.To test for agonist activity of the IgGs, cells were stimulated withIgGs (titrations 1:3 starting from 100 nM) or with the known agonistGLP1 (7-36) peptide (titrations 1:6 starting from 12.5 nM) for 30minutes at 37° C. To test for allosteric activity of the IgGs, cellswere incubated with IgGs at a fixed concentration of 100 nM for 1 hourat room temperature to allow binding, followed by stimulation with GLP1(7-36) peptide (titrations 1:6 starting from 12.5 nM) for 30 minutes at37° C. Intracellular cAMP levels were detected by following the assaykit instructions.

As seen in FIGS. 17A-17B, none of the IgGs initiated an agonist signal.GLP1R-241 was also tested for cAMP allosteric effect (FIG. 17C),beta-arrestin recruitment (FIG. 17D), and internalization (FIG. 17E).Several of the IgGs acted as negative allosteric modulators by changingthe signaling response of these cells to GLP1 (7-36) in an inhibitorymanner as seen in FIGS. 18A-18B. Table 18 shows the EC50 (nM) valuescorresponding to FIG. 18A and Table 19 shows the EC50 corresponding toFIG. 18B.

TABLE 18 EC50 (nM) Values + no Ab +GLP1R-238 +GLP1R-239 +GLP1R-240+GLP1R-241 +GLP1R-242 GLP1R-243 GLP1R-244 EC50 0.05946 0.08793 0.079950.06539 0.1027 ~0.06532 0.1282 0.1536

TABLE 19 EC50 (nM) Values pGPCR- pGPCR- +no Ab 43-GLP1R 44-GLP1R EC500.05946 2.948 3.485

The data shows pharmacological and functional effects of GLP1Rmodulators.

Example 13: GLP1R Agonists and Antagonists

This example illustrates identification of GLP1R agonists andantagonists.

Experiments were performed similarly to Example 12. Six GLP1Rimmunoglobulins (IgGs) were assayed for binding and functional assays todetermine which clones were agonists or antagonists. The GLP1R IgGstested included GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3,GLP1R-241, and GLP1R-2. GLP1R-241, GLP1R-3, and GLP1R-2 were previouslydescribed in Examples 10 and 12. Heavy chain sequences for GLP1R-59-2,GLP1R-59-241, GLP1R-59-243, GLP1R-43-8, and GLP1R-3 is seen in Table 20.

TABLE 20 Variable Heavy Chain Sequences SEQ ID NO. VariantVariable Heavy Chain Sequence 2317 GLP1R-59-2QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAVISYDAGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFETVVEGIQWYEALKAGKLGEVVPADDAFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2318 GLP1R-59-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQAPGQGLEWMGGIIPIFGTTN 241YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHMSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2319 GLP1R-59-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGINWVRQAPGQGLEWMGGIIPIFGTAN 243YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG 2320GLP1R-3 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSFISYDESNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2321GLP1R-43-8 MEWSWVFLFFLSVTTGVHSEVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADSAKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPHNDDRYDWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The GLP1R IgGs were characterized for thermal ramp stability (Tm andTagg). The UNcle platform was used to characterize the IgGs and the datais seen in Table 21.

TABLE 21 Thermal Ramp Stability Measurements Average Average Average %CV SD Tm1 % CV Tm2 % CV Tagg 266 Tagg Tagg Sample (° C.) Tm1 SD Tm1 (°C.) Tm2 SD Tm2 (° C.) 266 266 GLP1R-59-2 60.6 0.08 0.05 84.6 0.71 0.658.3 0.29 0.17 GLP1R-59-241 66 6.52 4.3 73.6 0.41 0.3 57.8 0.69 0.4GLP1R-59-243 60.9 0.33 0.2 75.2 0.8 0.6 55.9 0.72 0.4 GLP1R-3 66.7 0.60.4 73.5 0.54 0.4 68.4 0.58 0.4 GLP1R-241 68.2 0.82 0.56 75.7 0.94 0.7165.9 0.76 0.5 GLP1R-2 61.8 1.17 0.72 74.8 1.27 0.95 60.5 0.12 0.07

The GLP1R IgGs were then assayed in binding assays coupled to flowcytometry analysis using similar methods as described in Example 12.Briefly, stably expressing Flag-GLP1R-GFP CHO cells or CHO-parent cellswere incubated with primary IgG (100 nM or 1:3 titrations). Secondaryantibody incubation involved Alexa 647 conjugated goat-anti-human IgG.Flow cytometry measured the GFP signal against the Alexa 647 signal toidentify IgGs that specifically bound to the target (GLP1R). Ligandcompetition assays involved co-incubating the primary IgG with 1 μM GLP1(7-36). Data for GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3,GLP1R-241, and GLP1R-2 are seen in FIGS. 19A-19F.

Functional assays were also performed using the GLP1R IgGs using similarmethods as described in Example 12. Briefly, cAMP, beta-arrestinrecruitment and activated receptor internalization assays were obtainedfrom Eurofins DiscoverX and utilized untagged GLP-1R overexpressingCHO-K1 or U2OS cells. These were used to test for either agonistactivity of the IgGs as compared with GLP1 (7-36) or for antagonisticactivity of the IgGs by pre-incubating cells with IgGs and examiningtheir effects on GLP1 (7-36)-induced signaling. For the cAMP assays,following GLP1 (7-36) or IgG stimulation, the cellular cAMP levels aremeasured using a homogenous, no wash, gain-of-signal competitiveimmunoassay based on Enzyme Fragment Complementation (EFC) technology.Data from the functional assays for GLP1R-59-2, GLP1R-59-241,GLP1R-59-243, GLP1R-3, GLP1R-241, and GLP1R-2 is seen in FIGS. 20A-20F.The EC50 (nM) data for GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3,GLP1R-241, and GLP1R-2 is seen in Tables 22-23. As seen in Table 23, theEC50 data for GLP1R-3 showed a 2.2-fold difference. The EC50 data forGLP1-241 showed a 1.7-fold difference. The EC50 data for GLP1R-2 showeda 0.8-fold difference.

TABLE 22 EC50 (nM) for GLP1R-59-2, GLP1R-59-241, and GLP1R-59-243 GLP1RIgG EC50 GLP1 (7-36) EC50 GLP1R-59-2 0.842 0.4503 GLP1R-59-241 0.72230.4731 GLP1R-59-243 0.8209 0.4731

TABLE 23 EC50 (nM) for GLP1R-3, GLP1R-241, and GLP1R-2 GLP1R IgG (+100nM) EC50 No Antibody EC50 GLP1R-3 1.311 0.6053 GLP1R-241 0.1027 0.05946GLP1R-2 0.07947 0.1031

GLP1R-3 was also assayed to determine specificity of GLP1R versus GL1P2Rbinding and determined to be specific for GLP1R over GLP2R (data notshown). Binding of GLP1R-3, GLP1R-59-242, and GLP1R-43-8 on mouse,macaca, and human GLP1R was determined. GLP1R-3 at 100 nM, GLP1R-59-242at 100 nM, and GLP1R-43-8 at 100 nM were found to bind mouse, macaca,and human GLP1R (data not shown). GLP1R-3 was also found to bound humanpancreatic precursor cells expressing endogenous GLP1R.

Binding of GLP1R-59-2, GLP1R-59-241, and GLP1R-59-243 on mouse, macaca,and human GLP1R was determined. GLP1R-59-2 at 100 nM, GLP1R-59-241 at100 nM, and GLP1R-59-243 at 50 nM were found to bind mouse, macaca, andhuman GLP1R (data not shown). GLP1R-59-2 was also found to bound humanpancreatic precursor cells expressing endogenous GLP1R.

This example shows GLP1R IgGs with agonistic and antagonist properties.Several of the IgGs induced cAMP signaling, beta-arresting recruitment,and receptor internalization similar to GLP1 (7-36).

Example 14: VHH Libraries

Synthetic VHH libraries were developed. For the ‘VHH Ratio’ library withtailored CDR diversity, 2391 VHH sequences (iCAN database) were alignedusing Clustal Omega to determine the consensus at each position and theframework was derived from the consensus at each position. The CDRs ofall the 2391 sequences were analyzed for position-specific variation,and this diversity was introduced in the library design. For the ‘VHHShuffle’ library with shuffled CDR diversity, the iCAN database wasscanned for unique CDRs in the nanobody sequences. 1239 unique CDR1's,1600 unique CDR2's, and 1608 unique CDR3's were identified and theframework was derived from the consensus at each framework positionamongst the 2391 sequences in the iCAN database. Each of the uniqueCDR's was individually synthesized and shuffled in the consensusframework to generate a library with theoretical diversity of3.2×10{circumflex over ( )}9. The library was then cloned in thephagemid vector using restriction enzyme digest. For the ‘VHH hShuffle’library (a synthetic “human” VHH library with shuffled CDR diversity),the iCAN database was scanned for unique CDRs in the nanobody sequences.1239 unique CDR1's, 1600 unique CDR2's, and 1608 unique CDR3's wereidentified and framework 1, 3, and 4 was derived from the human germlineDP-47 framework. Framework 2 was derived from the consensus at eachframework position amongst the 2391 sequences in the iCAN database. Eachof the unique CDR's was individually synthesized and shuffled in thepartially humanized framework using the NUGE tool to generate a librarywith theoretical diversity of 3.2×10{circumflex over ( )}9. The librarywas then cloned in the phagemid vector using the NUGE tool.

The Carterra SPR system was used to assess binding affinity and affinitydistribution for VHH-Fc variants. VHH-Fc demonstrate a range ofaffinities for TIGIT, with a low end of 12 nM K_(D) and a high end of1685 nM K_(D) (data not shown). Table 23A provides specific values forthe VHH-Fc clones for ELISA, Protein A (mg/ml), and K_(D) (nM). FIG. 21Aand FIG. 21B depict TIGIT affinity distribution for the VHH libraries,over the 20-4000 affinity threshold (FIG. 21A; monovalent K_(D)) and the20-1000 affinity threshold (FIG. 21B; monovalent K_(D)). Out of the 140VHH binders tested, 51 variants had affinity <100 nM, and 90 variantshad affinity <200 nM.

TABLE 23A ELISA, Protein A, and K_(D) of VHH-Fc Clones ProA Clone ELISALibrary (mg/ml) K_(D) (nM) Variant 31-1 5.7 VHH hShuffle 0.29 12 Variant31-6 9.6 VHH hShuffle 0.29 14 Variant 31-26 5.1 VHH hShuffle 0.31 19Variant 30-30 8 VHH Shuffle 0.11 23 Variant 31-32 8 VHH hShuffle 0.25 27Variant 29-10 5 VHH Ratio 0.19 32 Variant 29-7 7.3 VHH Ratio 0.28 41Variant 30-43 13.5 VHH Shuffle 0.18 44 Variant 31-8 12.7 VHH hShuffle0.29 45 Variant 31-56 11.7 VHH hShuffle 0.26 46 Variant 30-52 4.2 VHHShuffle 0.22 49 Variant 31-47 8.8 VHH hShuffle 0.23 53 Variant 30-15 9.3VHH Shuffle 0.26 55 Variant 30-54 5.5 VHH Shuffle 0.3 58 Variant 30-4910.3 VHH Shuffle 0.26 62 Variant 29-22 3.4 VHH Ratio 0.27 65 Variant29-30 9.2 VHH Ratio 0.28 65 Variant 31-35 5.7 VHH hShuffle 0.24 66Variant 29-1 10.4 VHH Ratio 0.09 68 Variant 29-6 6.8 VHH Ratio 0.29 69Variant 31-34 6 VHH hShuffle 0.32 70 Variant 29-12 6.2 VHH Ratio 0.23 70Variant 30-1 5.4 VHH Shuffle 0.39 71 Variant 29-33 3.9 VHH Ratio 0.15 74Variant 30-20 4.6 VHH Shuffle 0.19 74 Variant 31-20 6.6 VHH hShuffle0.37 74 Variant 31-24 3.1 VHH hShuffle 0.15 75 Variant 30-14 9.9 VHHShuffle 0.19 75 Variant 30-53 7.6 VHH Shuffle 0.24 78 Variant 31-39 9.9VHH hShuffle 0.32 78 Variant 29-18 10.9 VHH Ratio 0.19 78 Variant 30-9 8VHH Shuffle 0.4 79 Variant 29-34 8.6 VHH Ratio 0.21 80 Variant 29-27 8.6VHH Ratio 0.18 82 Variant 29-20 5.9 VHH Ratio 0.26 83 Variant 30-55 6VHH Shuffle 0.41 85 Variant 30-39 6.1 VHH Shuffle 0.07 88 Variant 31-156.2 VHH hShuffle 0.32 88 Variant 29-21 4.3 VHH Ratio 0.23 88 Variant29-37 5.3 VHH Ratio 0.26 89 Variant 29-40 6.6 VHH Ratio 0.31 90 Variant31-30 3.2 VHH hShuffle 0.33 93 Variant 31-10 12.3 VHH hShuffle 0.31 94Variant 29-3 13.6 VHH Ratio 0.11 94 Variant 30-57 5.2 VHH Shuffle 0.2495 Variant 29-31 4.4 VHH Ratio 0.18 96 Variant 31-27 8.1 VHH hShuffle0.31 96 Variant 31-33 6 VHH hShuffle 0.32 96 Variant 30-40 7.1 VHHShuffle 0.21 99 Variant 31-18 4.1 VHH hShuffle 0.36 99 Variant 30-5 9.3VHH Shuffle 0.05 100

Example 15: VHH Libraries for GLP1R

A VHH library for GLP1R was developed similar to methods described inExample 14. Briefly, stable cell lines expressing GLP1R were generated,and target expression was confirmed by FACS. Cells expressing >80% ofthe target were then used for cell-based selections. Five rounds ofcell-based selections were carried out against cells stablyoverexpressing the target of interest. 10⁸ cells were used for eachround of selection. Before selection on target expressing cells, phagefrom each round was first depleted on 10⁸ CHO background cells.Stringency of selections was increased by increasing the number ofwashes in subsequent rounds of selections. The cells were then elutedfrom phage using trypsin, and the phage was amplified for the next roundof panning. A total of 1000 clones from round 4 and round 5 aresequenced by NGS to identify unique clones for reformatting as VHH-Fc.

53 out of the 156 unique GLP1R VHH Fc binders had a target cell meanfluorescence intensity (MFI) value that was 2-fold over parental cells.The data for variant GLP1R-43-77 is seen in FIGS. 22A-22B and Tables23B-24.

TABLE 23B Panning Summary VHH-Fc FACS binders Unique (MFI values 2-foldLibrary Phage over parental cells) VHH hShuffle 58  6 VHH Ratio/Shuffle98 47

TABLE 24 GLP1R-43-77 Data Subset Name with Gating Path CountMedian:RL1-A Sample E10.fcs/CHO-parent 11261  237 SampleE10.fcs/CHO-GLP1R 13684 23439

Example 16. GLP1R Libraries with Varied CDR's

A GLP1R library was created using a CDR randomization scheme.

Briefly, GLP1R libraries were designed based on GPCR antibody sequences.Over sixty different GPCR antibodies were analyzed and sequences fromthese GPCRs were modified using a CDR randomization scheme.

The heavy chain IGHV3-23 design is seen in FIG. 23A. As seen in FIG.23A, IGHV3-23 CDRH3's had four distinctive lengths: 23 amino acids, 21amino acids, 17 amino acids, and 12 amino acids, with each length havingits residue diversity. The ratio for the four lengths were thefollowing: 40% for the CDRH3 23 amino acids in length, 30% for the CDRH321 amino acids in length, 20% for the CDRH3 17 amino acids in length,and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity wasdetermined to be 9.3×10⁸, and the full heavy chain IGHV3-23 diversitywas 1.9×10¹³

The heavy chain IGHV1-69 design is seen in FIG. 23B. As seen in FIG.23B, IGHV1-69 CDRH3's had four distinctive lengths: 20 amino acids, 16amino acids, 15 amino acids, and 12 amino acids, with each length havingits residue diversity. The ratio for the four lengths were thefollowing: 40% for the CDRH3 20 amino acids in length, 30% for the CDRH316 amino acids in length, 20% for the CDRH3 15 amino acids in length,and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity wasdetermined to be 9×10⁷, and the full heavy chain IGHV-69 diversity is4.1×10¹².

The light chains IGKV 2-28 and IGLV 1-51 design is seen in FIG. 23C.Antibody light chain CDR sequences were analyzed for position-specificvariation. Two light chain frameworks were selected with fixed CDRlengths. The theoretical diversities were determined to be 13800 and5180 for kappa and light chains, respectively.

The final theoretical diversity was determined to be 4.7×10¹⁷ and thefinal, generated Fab library had a diversity of 6×10⁹. See FIG. 23D.

The purified GLP1R IgGs were assayed to determine cell-based affinitymeasurements and for functional analysis. FACS binding was measuredusing purified GLP1R IgG. As seen in FIG. 23E, the GLP1R IgG boundselectively to GLP1R-expressing cells with affinities in the lownanomolar range, demonstrating an IgG that selectively binds targetexpressing cell with an affinity of 1.1 nM. FACS binding was alsomeasured in GLP1R IgGs generated using methods described in Examples4-10. As seen in FIG. 23F, GLP1R IgGs bind selectively toGLP1R-expressing cells with affinities in the low nanomolar range.

cAMP assays using purified GLP1R IgG demonstrated that presence of GLP1RIgGs resulted in a left shift of the dose response curve of the GLP1agonist induced cAMP response in GLP1R expressing CHO cells as seen inFIG. 23G. GLP1R IgGs generated using methods described in Examples 4-10also resulted in a left shift of the dose response curve of the receptoragonist induced cAMP response in GLP1R expressing CHO cells (FIG. 23H).

The data shows the design and generation of GLP1R IgGs with improvedpotency and function.

Example 17. Oral Glucose Tolerance Mouse Model

The objective of this study was to evaluate the acute effects of achimeric antibody GLP1R agonist and antagonist on glycemic control in amouse model of diet induced obesity in C57BL/6J DIO mice. The testarticles are seen below in Table 25.

TABLE 25 Test Article Identification GLP1 Agonist Ab GLP1 Antagonist AbAb Control Positive Control Identification GLP1R-59-2 GLP1R-3 GLP1R-2Liraglutide Physical Clear Liquid Clear Liquid Clear Liquid DescriptionPurity 95% 95% TBD Concentration 2.7 mg/ml 3.7 mg/ml TBD StorageTemperature Temperature Temperature Temperature Conditions set tomaintain set to maintain set to maintain set to maintain 4° C. 4° C. 4°C. 4° C. Provided by Sponsor Sponsor Sponsor Testing Facility — = Notapplicable.

For each test article, 7 different test article groups were generated assummarized in the following Table 26 with 8 animals per group.

TABLE 26 Experimental Design Dose Dose Test Dose Level VolumeConcentration Dose Number of Group No. Material (mg/kg/day) (mL/kg)(mg/mL) Diet Regimen Route animals 1 GLP1R-2 0 5 0 HFD QD SC 8 2Liraglutide 0.2 5 0.04 HFD QD SC 8 3 GLP1R-2 10 5 2 HFD QD SC 8Liraglutide 0.2 5 0.04 4 GLP1R-59-2 10 5 2 HFD QD SC 8 5 GLP1R-59-2 10 52 HFD QD SC 8 Liraglutide 0.2 5 0.04 6 GLP1R-3 10 5 2 HFD QD SC 8 7GLP1R-3 10 5 2 HFD QD SC 8 Liraglutide 0.2 5 0.04 No. = Number; ; HFD =high fat diet; QD = once daily; SC = Subcutaneous injection

On Day 3 (all animals) and Day 1 (Group 1-7), a non-fasting bloodglucose was determined by tail snip. Approximately 5-10 μL of blood wascollected. The second drop of blood from the animal was placed on ablood glucose test strip and analyzed using a hand-held glucometer(Abbott Alpha Trak).

After a non-fasting blood glucose measurement was made on the day of theprocedure, the animals were weighed, tails marked, and the animalsplaced in clean cages without food. The animals were fasted for 4 hoursand a fasting blood glucose measurement was determined. The animals werethen treated with the indicated test article(s) as shown in Table 26.

The oral glucose tolerance test (OGTT) was administered to each animal60 minutes later. The animals were dosed via oral gavage with 2 g/kgglucose (10 mL/kg). Blood glucose was determined via tail snip with thesecond drop of blood from the animal placed on a hand-held glucometer(Abbott Alpha Trak) at the following times relative to the glucose dose:0 (just prior to glucose dose), 15, 30, 60, 90, and 120 minutes.Additional blood samples were obtained at the 15 minute and 60 minutetime points of the OGTT for estimation of serum insulin.

FIGS. 24A-24B show GLP1R-3 inhibits GLP1:GLP1R signaling (FIG. 24A) withcomplete inhibition at higher concentrations (FIG. 24B). As seen in FIG.24C, GLP1R-3 dosed animals maintained sustained high glucose levelsafter glucose administration, indicating GLP1:GLP1R signal blockade. Asseen in FIG. 24D, GLP1R-59-2 dose at 10 mg/kg exhibited a sustained, lowglucose levels similar to liraglutide control.

The data shows that the GLP1R antibodies generated have functionaleffects in a mouse model for glucose tolerance.

Example 18. GLP1R Agonists and Antagonists Effects in Wild-Type Mice

The effects of GLP1R-59-2 (agonist) and GLP1R-3 (antagonist) inwild-type mice were determined in this Example.

15 C57BL/6NHsd Mice were used and subjected to a Glucose Tolerance Test(GTT). The in vivo GTT test was performed on three groups of mice with 5mice per group. All three groups were fasted for 13.5 hours before beingweighed, time Zero Blood Glucose measured, and then injected i.p. with a30% dextrose solution at a dose of 10 uL/gram body weight. Blood glucosemeasurements were recorded for each mouse at 15, 30, 60, 120, and 180minutes after dextrose injection. A first group of mice were treatedwith GLP1R-59-2 at two doses: 10 mg/kg of GLP1R-59-2 at time of fasting(˜13.5 hrs. prior to GTT) and again two hours before start of GTT with10 mg/kg of GLP1R-59-2. A second group of mice were treated with GLP1R-3at two doses: 10 mg/kg of GLP1R-3 at time of fasting (˜13.5 hrs. priorto GTT) and again two hours before start of GTT with 10 mg/kg ofGLP1R-3. A third group of mice were the control mice and were nottreated. Data is seen for GLP1R-59-2 (agonist), GLP1R-3 (antagonist),and control in FIGS. 25A-25D. FIG. 25A shows the blood glucose levels inmice (y-axis) treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist),and control over time (in minutes, x-axis). FIG. 25B shows the bloodglucose levels in mice (y-axis) treated with GLP1R-59-2 (agonist),GLP1R-3 (antagonist), and control. As seen in FIG. 25C, a significantreduction in blood glucose was observed in GLP1R-59-2 (agonist) treatedmice in both the fasted (p=0.0008) and non-fasted (p<0.0001) micecompared to control. As seen in FIG. 25D, pre-dosed GLP1R-3 (antagonist)animals did not show decreased glucose in a 6 hour fast whereas controlmice exhibited a decrease.

Example 19. Exemplary Sequences

Exemplary sequences of GLP1R are seen in Table 27. Table 27. GLP1RSequences SEQ GLP1R Sequence ID NO: Variant

TABLE 27 GLP1R Sequences SEQ GLP1R ID NO: Variant Sequence 2411GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKEREFLAAITSGGATTYDD01 NRKSRFTISADNSKNTAYLQMNSLKPEDTAVYYCWAALDGYGGRWGQGTLVTVSS 2412GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFRINRMGWFRQAPGKEREWVSTICSRGDTYYADS02 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLDGYSGSWGQGTLVTVSS 2413 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGRDFRVKNMGWFRQAPGKEREFVARITWNGGSAYY 03ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARILSRNWGQGTLVTVSS 2414 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMGWFRQAPGKEREFVAAISSGGRTSYADS 04VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYEGSWGQGTLVTVSS 2415 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYAMGWFRQAPGKEREFVAAISSGGRTRYADN 05VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYNGIWGQGTLVTVSS 2416 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGHTSDTYIMGWFRQAPGKEREFVSLINWSSGKTIYAD 06SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKGDYRGGYYYPQTSQWGQGTLVTVSS 2417GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKEREFVATIPSGGSTYYADS07 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYNGSWGQGTLVTVSS 2418 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTFGEFTMGWFRQAPGKERERVATITSGGSTNYADS 08VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVDDYSGSWGQGTLVTVSS 2419 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREVVAGIAWGDGITYYA 09DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNWGQGTLVTVSS 2420 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGRTFSSGVMGWFRQAPGKEREFVAAINRSGSTFYADS 10VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDWGQGTLVTVSS 2421GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGVTLDDYAMGWFRQAPGKEREFVAAINRSGSITYYA11 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGTLV TVSS2422 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGLTFGIYAMGWFRQAPGKEREFVATISRSGASTYYAD 12SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYNDYDRGHDWGQGTLVTVSS 2423GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSDGMGWFRQAPGKERELVAAINRSGSTFYADS13 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTARPGIFTTAPVEDWGQGTLVTVSS 2424GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTCGNYTMGWFRQAPGKERESVASITSGGRTNYADS14 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLDGYTGSWGQGTLVTVSS 2425 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTFNYYPMGWFRQAPGKEREWVATISRGGGTYYAD 15NVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYSGIWGQGTLVTVSS 2426 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTMGWFRQAPGKEREFVGFITGSGGTTYYADS 16VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAARRYGNLYNTNNYDWGQGTLVTVSS 2427GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQAPGKEREFVAAISWRGGSTNYAD17 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATLGEPLVKYTWGQGTLVTVSS 2428GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGISSKGGSSTYYA18 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTLVTV SS 2429GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGSRFSGRFNILNMGWFRQAPGKEREFVAAISRSGDTTY 19YADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASLRNSGSNVEGRWGQGTLVTVS S 2430GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGGTSNSYRMGWFRQAPGKEREFVAVISWTGGSTYYA20 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVALDGYSGSWGQGTLVTVSS 2431GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFNIGTYTMGWFRQAPGKEREFVAAIGSNGLANYAD21 NVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYSGTWGQGTLVTVSS 2432GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSVYAMGWFRQAPGKEREFVAGIHSDGSTLYADS22 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYMGTWGQGTLVTVSS 2433 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVMGWFRQAPGKERELVAAVRWSGGITWYA 23DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVYYGDWEGSEPVQHEYDWGQGT LVTVSS 2434GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMGWFRQAPGKEREFVAAIYCSDGSTQYA24 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAEALDGYWGQGTLVTVSS 2435 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGYTFRAYAMGWFRQAPGKEREMVAAMRWSGGITWY 25ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDGLPIKYDWGQGTLV TVSS 2436GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKERECVTAIFSDGGTYYADN26 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYNGYWGQGTLVTVSS 2437 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGIHFAISTMGWFRQAPGKEREIVTAINWSGARTYYAD 27SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKFVNTDSTWSRSEMYTWGQGTLVTV SS 2438GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTFTSYAMGWFRQAPGKEREGVAVIDSDGTTYYAD28 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYLDGYSGSWGQGTLVTVSS 2439GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSLPMGWFRQAPGKERELVAIRWSGGSTVYADS29 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWGQGTLVTVSS 2440GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSGVMGWFRQAPGKEREFVAAINRSGSTFYADS30 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTWGQGTLVTVSS 2441GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWFRQAPGKERELVAAISSGGSTSYADS31 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAMDGYSGSWGQGTLVTVSS 2442 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREYVAAISGSGSITNYAD 32SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANGIESYGWGNRHFNWGQGTLVTVSS 2443GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVAAIRWSGGITWYA33 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAIFDVTDYERADWGQGTLVTVSS 2444GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFAFSGYAMGWFRQAPGKEREFVAAISWSGGITWYA34 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAFVTTNSDYDLGRDWGQGTLVTVSS 2445GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGIPASIRTMGWFRQAPGKEREGVSWISSSDGSIYYADS 35VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVAALDGYSGSWGQGTLVTVSS 2446 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGRTFSSLPMGWFRQAPGKERELVAIRWSGGSTVYADS 36VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTLVTVSS 2447 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSWISTTDGSTYYA 37DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGIWGQGTLVTVSS 2448 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYAMGWFRQAPGKEREFVTAIDSESRTLYADS 38VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALLDGYLGTWGQGTLVTVSS 2449 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMGWFRQAPGKEREFLGSILWSDDSTNYAD 39SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANLKQGSYGYRFNDWGQGTLVTVSS 2450GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGTIVNIHVMGWFRQAPGKERELVAAITSGGSTSYADN40 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRHFEYDWGQGTLVTVSS 2451GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRSLGTYHMGWFRQAPGKEREGVSWISSSDGSTYYA41 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVLDGYSGSWGQGTLVTVSS 2452GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDTGMGWFRQAPGKEREFVAAIRWSGKETWYA42 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEDPSMYYTLEEYEYDWGQGTLVTV SS 2453GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMGWFRQAPGKERECVAAISSSDGRTYYAD43 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGNWGQGTLVTVSS 2454GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKEREFIATIFSGGDTDYADSV 44KGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTLVTVSS 2455 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKEREIVASITSGGRKNYADS 45VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDDYSGSWGQGTLVTVSS 2456 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGHSFGNFPMGWFRQAPGKEREVIAAIDWSGGSTFYAD 46SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGIGVYGWGQGTLVTVSS 2457 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGSSFRFRAMGWFRQAPGKEREFVAAINRGGKISHYAD 47SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYIRPDTYLSRDYRKYDWGQGTLVTV SS 2458GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREGVAAIDSDGRTRYA48 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTLVTVSS 2459GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGNILSLNTMGWFRQAPGKEREFVAGISWSGGSTYYAD49 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTLVTVSS 2460GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGITFRRYDMGWFRQAPGKEREGVAYISSSDGSTYYAD50 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDDYSGGWGQGTLVTVSS 2461GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTLSNYAMGWFRQAPGKEREFVAAISRSGSSTYYAD51 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEMSGISGWDWGQGTLVTVSS 2462GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGYTTSINTMGWFRQAPGKEREVVAAISRTGGSTYYAD52 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRRFEYDWGQGTLVTVSS 2463GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAMKPDGSITYYADS53 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLELFHDEYNWGQGTLVTV SS 2464GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSIFSLNAMGWFRQAPGKERELVAGISSKGGSTYYAD54 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTLVTVSS 2465GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYRMGWFRQAPGKEREAVAAIASMGGLTYYA55 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYIGSWGQGTLVTVSS 2466GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFGAFTMGWFRQAPGKERERVAAITCSGSTTYADS56 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYNGSWGQGTLVTVSS 2467 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGIPSTIRAMGWFRQAPGKERESVGRIYWRDDNTYYAD 57SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTLVTVSS 2468 GLP1R-40-EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREVVAGIAWGDGITYYA 58DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNYYYPISRDEYDWGQGT LVTVSS2469 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTIVPYTMGWFRQAPGKEREVVASISWSGKSTYYA 1DSVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAQRRWSQDWGQGTQVTVSS 2470 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 2DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTGRGERDYWGQGTQVTVSS 2471GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFSNYAMGWFRQAPGKEREFVATITWSGSSTYYA3 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYREYGYWGQGTQVTVSS 2472GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSIFHINPMGWFRQAPGKEREfVAAINIFGTTNYADSV 4KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVDGGPLWDDGYDWGQGTQVTVSS 2473 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVASINIFGTTKYADSV 5KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAVGWGPHNDDRYDWGQGTQVTVSS 2474 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGTTFSIYAMEWFRQAPGKERELVATISRSGGTTYYAD 6SVGGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAASWYYRDDYWGQGTQVTVSS 2475 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADS 7VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAVRWGPHNDDRYDWGQGTQVTVSS 2476GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADS8 AKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPHNDDRYDWGQGTQVTVSS 2477GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFILYGYAMGWFRQAPGKEREGVSSISPSDASTYYAD9 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLNTYSDSWGQGTQVTVSS 2478 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREGVTAISTSDGSTYYAD 10SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARDGYSGSWGQGTQVTVSS 2479 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGYTITNSYRMGWFRQAPGKEREFVAGITMSGFNTRY 11ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANRGLAGPAWGQGTQVTVSS 2480 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFTFDDNAMGWFRQAPGKEREFVSGISTSGSTTYYAD 12SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAAGGYDYWGQGTQVTVSS 2481 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSYYHMGWFRQAPGKEREGVSWISSYYSSTYYA 13DSESGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGYSCSWGQGTQVTVSS 2482 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSPFRLYTMGWFRQAPGKEREVVAHIYSYGSINYADS 14VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALWGHSGDWGQGTQVTVSS 2483 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSTFDTYGMGWFRQAPGKEREFVASITWSGSSTYYA 15DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANRIHWSGFYYWGQGTQVTVSS 2484GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTSSPYTMGWFRQAPGKEREFVSAISWSGGSTVYAD16 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALIRRAPYSRLETWGQGTQVTVSS 2485GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFPINAMGWFRQAPGKEREGVAAITNFGTTKYADS17 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDHYDWGQGTQVTVSS 2486GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFDTYAMGWFRQAPGKEREFVAAITWGGGRTYY18 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYRDYDYWGQGTQVTVSS 2487GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRRFSAYGMGWFRQAPGKEREFVAAVSWDGRNTYY19 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASTDDYGVDWGQGTQVTVSS 2488GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFDNYAMGWFRQAPGKEREFVSAISGDGGTTYYA20 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYRNRDYWGQGTQVTVSS 2489GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVSWITSFDASTYYAD21 SVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTQVTVSS 2490GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVSTISTGGSSTYYAD22 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTGRGRRDWGQGTQVTVSS 2491GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA23 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPVVPNTKDYWGQGTQVTVSS 2492GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNVFMIKDMGWFRQAPGKEREWVTAISWNGGSTDY24 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTQVTVSS 2493GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFPFSIWPMGWFRQAPGKEREFIATIFSGGDTDYADSV 25KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTQVTVSS 2494 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRGFSRYAMGWFRQAPGKEREFVAAIRWSGKETWY 26ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALGPVRRSRLEWGQGTQVTVSS 2495GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTSDIYGMGWFRQAPGKEREFVARIYWSSGNTYYA27 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAYRFSDYSRPAGYDWGQGTQVTV SS 2496GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNDFSFNSMGWFRQAPGKEREFLASVSWGFGSTYYA28 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCARAYGNPTWGQGTQVTVSS 2497 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFTDYPMGWFRQAPGKERELESFVPINGTSTYYAD 29SDSGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSCSWGQGTQVTVSS 2498 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVATISRGGSTTYYAD 30SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAGPRSGKDYWGQGTQVTVSS 2499 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFIFQLYVMGWFRQAPGKEREGVTYINNIDGSTYYAY 31SVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRDGYSGSWGQGTQVTVSS 2500 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYAMEWFRQAPGKERELVATISRSGGRTYYAD 32SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAANWYYRYDYWGQGTQVTVSS 2501 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFPFRINAMGWFRQAPGKERELVTAISSSGSSTYYADS 33VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASGYYATYYGERDYWGQGTQVTVSS 2502GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTLSSYTMGWFRQAPGKEREFVSAISRGGGNTYYAD34 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSYAEYDYWGQGTQVTVSS 2503GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYGMGWFRQAPGKEREGVAAINGGGDSTNYA35 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAASASPYSGRNYWGQGTQVTVSS 2504GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGLtfSTTVMGWFRQAPGKEREGDGYISITDGSTYYADS 36VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAALDGYSGSWGQGTQVTVSS 2505 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTLENYRMGWFRQAPGKEREFVAAVSWSSGNAYY 37ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANWKMLLGVENDWGQGTQVTVS S 2506GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA38 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTVYGERDYWGQGTQVTVSS 2507GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSILSISPMGWFRQAPGKERELVAINFSWGTTDYADSv 39KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAYEQGVYRWDWGQGTQVTVSS 2508 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 40DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAERYRYSGYYARDSWGQGTQVTVS S 2509GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTLSDYAMGWFRQAPGKEREFVSAISRDGTTTYYA41 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTSQYATDYWGQGTQVTVSS 2510GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRDLDYYVMGWFRQAPGKERELVAIKFSGGTTDYAD42 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCADIAYEEGVYRWDWGQGTQVTVSS 2511GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFTFNAMGWFRQAPGKEREFVAGITRSAVSTSYAD43 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTQVTVSS 2512GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFDSYAMGWFRQAPGKEREFVAAITSSGGNTYYA44 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPARYGARDYWGQGTQVTVSS 2513GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFNNDHMGWFRQAPGKEREFVAVIEIGGATNYAD45 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATWDGRQVWGQGTQVTVSS 2514 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGGTFRKLAMGWFRQAPGKERELVAAIRWSGGITWYA 46DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATLAKGGGRWGQGTQVTVSS 2515 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 47DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRAPSDRDYWGQGTQVTVSS 2516GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFRIYAMGWFRQAPGKERELVSSISWNSGSTYYAD48 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAAYSYTQGTTYESWGQGTQVTVSS 2517GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFTSYRMGWFRQAPGKEREWMGTIDYSGRTYYA49 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAMDGYSGSWGQGTQVTVSS 2518GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAAINWNGDTTYYA50 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRYSDYDYWGQGTQVTVSS 2519GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRFFSTRVMGWFRQAPGKERELVAIKFSGGTTDYADS51 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTQVTVSS 2520GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA52 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSVYGTRDYWGQGTQVTVSS 2521GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSIDVMGWFRQAPGKEREGVSYISMSDGRTYYAD53 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAELDGYSGSWGQGTQVTVSS 2522GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGLSFSGYTMGWFRQAPGKEREVVAAISRTGGSTYYA54 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALIQRRAPYSRLETWGQGTQVTVSS 2523GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTLSIYGMGWFRQAPGKEREGVAAISWSDGSTSYAD55 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVADIGLASDFDYWGQGTQVTVSS 2524GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYAMGWFRQAPGKEREFVATITRSSGNTYYAD56 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPFKPYSYDYWGQGTQVTVSS 2525GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSTFSIYTMGWFRQAPGKEREFVAAISGSSDSTYYADS 57VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATVPKTRYTRDYWGQGTQVTVSS 2526 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGNTFSSYAMGWFRQAPGKEREFVAIISRSGGRTYYAD 58SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAPYNETNSWGQGTQVTVSS 2527 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSTFSTYAMGWFRQAPGKEREFVASISRSGGRTYYAD 59SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARYNERNSWGQGTQVTVSS 2528 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGGTLNNNPMAMGWFRQAPGKEREFVVAIYWSNGKT 60PYADSVKRRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGAWGQGTQVTVSS 2529GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA61 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRAPSERDYWGQGTQVTVSS 2530GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFNNNDMGWFRQAPGKEREFVAVIKLGGATTYDD62 YSEGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATWDARHVWGQGTQVTVSS 2531 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRAFSYYNMGWFRQAPGKEREGVSWISSSDGSTYYA 63DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGCSGSWGQGTQVTVSS 2532 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSTFSTYAMGWFRQAPGKEREFVAAINRSGASTYYA 64DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALLGGRGGCGKGYWGQGTQVTVS S 2533GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSILDTYAMGWFRQAPGKERELVSGINTSGDTTYYAD65 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLAGYEYWGQGTQVTVSS 2534 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSTLSINAMGWFRQAPGKEREFVAHMSHDGTTNYAD 66SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCARLPNYRWGQGTQVTVSS 2535 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSIFRLNAMGWFRQAPGKEREGVAAINNFDTTKYAD 67SSKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRSDDRWGQGTQVTVSS 2536 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGLTNPPFDNFPMGWFRQAPGKEREFVAVISWTGGSTY 68YAPSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCPAVYPRYYGDDDRPPVDWGQGTQ VTVSS 2537GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGPTFSKAVMGWFRQAPGKEREFVAAMNWSGRSTYY69 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATPAGRGGYWGQGTQVTVSS 2538GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFSDYAMGWFRQAPGKEREFVATINWGGGRTYYA70 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYARDYWGQGTQVTVSS 2539GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFILSDYAMGWFRQAPGKEREFVAAISSSEASTYYAD71 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRFWAGYDSWGQGTQVTVSS 2540GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGYTDYKYDMGWFRQAPGKEREFVAAISWGGGLTVY72 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVATVTDYTGTYSDGWGQGTQVT VSS 2541GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVATINWGGGNTYY73 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYAYDYWGQGTQVTVSS 2542GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYYMGWFRQAPGKERELVAVILRGGSTNYAD74 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARRYGNLYNTNNYDWGQGTQVTVS S 2543GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGSILSSYVMGWFRQAPGKEREFVSAISRSGTSTYYADS 75VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYDRDYWGQGTQVTVSS 2544 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFTLDNYAMGWFRQAPGKEREFVAAISWSGGSTYYA 76DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYSYDYWGQGTQVTVSS 2545GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNTYSYKVMGWFRQAPGKEREFVGIIIRNGDTTYYAD77 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASPKYMTAYERSYDWGQGTQVTVSS 2546GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRNYAMGWFRQAPGKEREFVATITTSGGNTYYAD78 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYRRDWGQGTQVTVSS 2547GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTMGWFRQAPGKEREVVAAITGSGRSTYYA79 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASAIGSGALRRFEYDWGQGTQVTVS S 2548GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTFSAYAMGWFRQAPGKEREGVAAIRWDGGYTRY80 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATTPTTSYLPRSERQYEWGQGTQV TVSS2549 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 81DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSVYGERDYWGQGTQVTVSS 2550GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGISQSGGSTAYAD82 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTQVTVS S 2551GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREMVASITSRKIPKYADS83 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVWSGRDWGQGTQVTVSS 2552 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFTFRRYVMGWFRQAPGKEREFVAAISRDGDRTYYA 84DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASTRLAGRWYRDSEYKWGQGTQVTV SS 2553GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSDNAMGWFRQAPGKEREFVATISRGGSRTSYAD85 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAGPRSGRDYWGQGTQVTVSS 2554GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFRSYAMGWFRQAPGKEREFVATITRNGDNTYYA86 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATVGTRYNYWGQGTQVTVSS 2555GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYVMGWFRQAPGKERELISGITWNGDTTYYA87 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVVRLGGYDYWGQGTQVTVSS 2556GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGIISNYHMGWFRQAPGKEREFVATITRSGGSTYYAD88 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAMAGRGRWGQGTQVTVSS 2557 GLP1R-43-EVQLVESGGGLVQAGGSLRLSCAASGFSFDDDYVMGWFRQAPGKERELVSAIGWSGASTYY 89ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGT QVTVSS 2558GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFPIYAMGWFRQAPGKEREWVSGISSRDDTTYYAD90 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAHRIVFRGTSVGDWRWGQGTQVTVSS 2559GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRAFSYYNMGWFRQAPGKEREGVSWISSSDGSTYYA91 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTQVTVSS 2560GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSIDVMGWFRQAPGKERELVAATGRRGGPTYYA92 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAARTSYSGTYDYGVDWGQGTQVTVS S 2561GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAINWSGSITYYA93 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVGRSGRDYWGQGTQVTVSS 2562GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADS94 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDRYDWGQGTQVTVSS 2563GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTLNNNPMAMGWFRQAPGKEREFVVAIYWSNGKT95 QYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTQVTVSS 2564GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFNNDHMGWFRQAPGKEREFVAVIEIGGATNYAD96 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASWDGRQVWGQGTQVTVSS 2565 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFAMGWMGWFRQAPGKEREFVARVSWDGRNAY 01YANSRFGRFTISADNSKNTAYLQMNSLKPEDTAVYYCPRYVSPARDHGCWGQGTLVTVSS 2566GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGLTISTYIMGWFRQAPGKEREFVAVVNWNGDSTYYA02 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGTLV TVSS2567 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWFRQAPGKERELVAAINRGGKITHYAD 03SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASLRNSGSNVEGRWGQGTLVTVSS 2568GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREFVAAISPSAVTTYYA04 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYDYYSDYPLPDANEYEWGQGTLVT VSS2569 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYIMGWFRQAPGKEREFVAVINRSGSTTYYAD 05SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYSNSSDYYSQEGAYDWGQGTL VTVSS 2570GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYVMGWFRQAPGKEREGVSYISSSDGRTHYAD06 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYNGSWGQGTLVTVSS 2571GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSRFGMGWFRQAPGKEREGVAAIGSDGSTSYADS07 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASGRDRYARDLSEYEYVWGQGTLVTVSS 2572GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFRFNAMGWFRQAPGKEREFVAAINWRGSHPYYA08 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATLGEPLVKYTWGQGTLVTVSS 2573GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREFLASVTWGFGSTYYA09 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATTTRSYDDTYRNSWVYNWGQGTL VTVSS2574 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYAMGWFRQAPGKERELVAAIRWSGGITWYA 10DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSGSDYLPMDWGQGTLVTVSS 2575GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGPTFTIYAMGWFRQAPGKEREFVGAISMSGEDTIYADS 11EKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYTSNTNYYNQEGAYDWGQGTLV TVSS 2576GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGPTFSNYYVGWFRQAPGKEREFVAAILCSGGITCYAD12 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYIGTWGQGTLVTVSS 2577GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFSSIGMGWFRQAPGKEREGVAAIGSDGSTSYADS13 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASDRYARVLTEYEYVWGQGTLVTVSS 2578GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGVTFNNYGMGWFRQAPGKERELVAAIRWSGSATFYA14 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADDGARGSWGQGTLVTVSS 2579GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFTMDGMGWFRQAPGKEREGVAAIGSDGSTSYAD15 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGSNIGGSRWRYDWGQGTLVTVSS 2580GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAAISPAALTTYYAD16 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYLPSPYYSSYYDSTKYEWGQGTLVT VSS2581 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSGFSPNVMGWFRQAPGKEREVVAAISWNGGSTYYA 17DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRRFEYDWGQGTLVTVSS 2582GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFGFYAMGWFRQAPGKERELVAAISWSDASTYYA18 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDYYNVSEYDWGQGTLV TVSS2583 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYPMGWFRQAPGKERECVSTIWSRGDTYYADN 19VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSATWGQGTLVTVSS 2584 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAMGWFRQAPGKERELVAAISWSNDITYYA 20DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDYYSVSEYDWGQGTLVT VSS 2585GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYTMGWFRQAPGKEREFVAGIYNDGTASYYA21 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFDGYTGNDWGQGTLVTVSS 2586GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREWVARMYLDGDYPYY22 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTLVTVSS 2587GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTISRYIMGWFRQAPGKERELVAAINRSGKSTYYAD23 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASTRFAGRWYRDSEYKWGQGTLVTVSS 2588GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSVYAMGWFRQAPGKEREFVAAVRWSGGITWY24 VDSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFDGYSGSDWGQGTLVTVSS 2589GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSIFSITEMGWFRQAPGKERELVAAIAVGGGITWYADS 25VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHDVDDDESPYYSGGYYRALYDWGQG TLVTVSS2590 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSIYSLDAMGWFRQAPGKERELVAAISPAALTTYYAD 26SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASMSLRPLDPASYSPDIQPYDWGQGTL VTVSS2591 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKERESVAAIDSDGRTHYAD 27SVISRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGDWGQGTLVTVSS 2592 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTLSfYAMGWFRQAPGKEREFVAAINRGGRISHYAD 28SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRRYGSPPHDGSSYEWGQGTLVTVS S 2593GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKEREFVAGISWTGGITYYA29 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNVGFEWGQGTLVTVSS 2594 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMGWFRQAPGKEREGVAAIGSDGSTSYAD 30SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLRATITNFDEYVWGQGTLVTVSS 2595GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFNRYPMGWFRQAPGKEREFVAHMSHDGTTNYA31 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAPGTRYYGSNQVNYNWGQGTLVTV SS 2596GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSIFSFNAMGWFRQAPGKEREFVAGITRRGLSTSYADS 32VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGIGVYGWGQGTLVTVSS 2597 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGGSISSINAMGWFRQAPGKERELVAGIITSGDSTYYAD 33SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGSAYVAGVRRRNAYHWGQGTLVTV SS 2598GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGGTFSADVMGWFRQAPGKEREFVAAISTGSITIYADSV 34KGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATYGYDSGLYFITDSNDYEWGQGTLVTVSS 2599GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVAAMRWRGGITWY35 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGTLYDDYDGLPIKYDWGQGTLV TVSS2600 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGDIFNINAMGWFRQAPGKEREPVAAISPAALTTYYAD 36SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATPIERLGLDAYEYDWGQGTLVTVSS 2601GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYNMGWFRQAPGKEREFVAAINWSGGITWYA37 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKWGQGTLV TVSS2602 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSISVFDAMGWFRQAPGKERELVAGISGSGGDTYYAD 38SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASPKYSTHSIFDASPYNWGQGTLVTVS S 2603GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTSDDYAMGWFRQAPGKEREFVAALRWSSSNIDYT39 YYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGHGDVSEYEYDWGQGTL VTVSS2604 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTFSPNVMGWFRQAPGKEREFVAAITSSGETTWYAD 40SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDWGQGTLVTVSS 2605GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVAAINWSGDNTHY41 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANWKMLLGVENDWGQGTLVTVSS 2606GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGDTFNCYAMGWFRQAPGKEREFVAVINWSGDNTHY42 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGRYDWGQGT LVTVSS2607 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSISTINVMGWFRQAPGKEREFVAAISPSAVTTYYADS 43VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGRGDVSEYEYDWGQGTLVTVSS 2608GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSKYRMGWFRQAPGKEREFVAAIRWSGGITWYA44 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIPHGIAGRITWGQGTLVTVSS 2609GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYAMGWFRQAPGKERELVAGIDQSGGITWYA45 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADDYLGGDNWYLGPYDWGQGTLVT VSS 2610GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTIDDYAMGWFRQAPGKEREFVAAVSGTGTIAYYA46 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYIDYDEALEETRGSYDWGQGTLV TVSS2611 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYVMGWFRQAPGKERELVAGITSGRDITYYA 47DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADGVLATTLNWDWGQGTLVTVSS 2612GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSGISFNAMGWFRQAPGKERELVAAISRSGDTTYYAD48 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLTTWADGPYRWGQGTLVTVSS 2613GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAAINRGGKISHYAD49 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRRYGNPPHDGSSYEWGQGTLVTVS S 2614GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYGMGWFRQAPGKERELVAIKFSGGTTDYADS50 vkGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWGQGTLVTVSS 2615GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAGISGSGGDTYYAD51 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTLVTVSS 2616GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAAINRGGKISHYAD52 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRRYGSPPHDGSSYEWGQGTLVTVS S 2617GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSDFSLNAMGWFRQAPGKEREFVAAISWSGGSTLYA53 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASNESDAYNWGQGTLVTVSS 2618GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLVNYDMGWFRQAPGKEREFVAAIRWSGGITWYA54 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMLPPWGQGTLVTVSS 2619GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFEKDAMGWFRQAPGKEREMVAAIRWSGGITCYA55 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSLPDDYDGLECEYDWGQGTLVT VSS2620 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSFFKINAMGWFRQAPGKEREFVAGITRSGGSTYYAD 56SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAESLGRWWGQGTLVTVSS 2621 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIRWSGGITWYAD 57SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASHDSDWGQGTLVTVSS 2622 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIRWSGGITWYAD 58SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASHDSDYGGTNANLYDWGQGTLVTV SS 2623GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTDRSNVMGWFRQAPGKEREFVAAINRSGSTFYADS59 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDWGQGTLVTVSS 2624GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSFFSINVMGWFRQAPGKERELVAATGRRGGPTYYA60 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTLVTV SS 2625GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREGVAAIDSDGRTRYA61 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGNWGQGTLVTVSS 2626GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNIFSLNTMGWFRQAPGKEREFVAAINCSGNHPYYAD62 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDDDGRDNWGQGTLVTVSS 2627GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAVSGSGDDTYYAD63 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYSSSSDYYSQEGAYDWGQGTLV TVSS2628 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTFPAYVMGWFRQAPGKERELLAVITRDGSTHYADS 64VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNGRWRIWSSRNPWGQGTLVTVSS 2629GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKERELVAVIGWGGKETW65 YADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEDPSMGYYTLEEYEYDWGQGT LVTVSS2630 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISMSGDDTAYA 66DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLRGRGDVSEYEYDWGQGTLVTVS S 2631GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVGAITWGGGNTYYA67 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTDGDYDGWGQGTLVTVSS 2632GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNTFSINVMGWFRQAPGKEREFVAAINWNGGSTDYA68 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLDNDWGQGTLVTVSS 2633GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSTHWMGWFRQAPGKEREVVAVIYTSDGSTYYA69 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANEYGLGSSIYAYKWGQGTLVTVSS 2634GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSISAMGWFRQAPGKEREFVAAISRSGGTTYYAD70 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDEDYALGPNEYDWGQGTLVTVSS 2635GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSTFRINAMGWFRQAPGKERELVAAISPAALTTYYAD71 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPYGSGSLYDDYDGLPIKYDWGQGT LVTVSS2636 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVAAISWSNDITYYAD 72SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALSEVWRGSENLREGYDWGQGTLVT VSS 2637GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGLPVDYYAMGWFRQAPGKERELVAAISGSGDSTYYA73 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQTEDSASIFGYGMDWGQGTLVTVS S 2638GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSTVNMGWFRQAPGKEREFVGAISRSGETTWYA74 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVDCPDYYSDYECPLEWGQGTLVTVS S 2639GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYAMGWFRQAPGKERELVAAVRWSGGITWY75 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGDTGGAAYGWGQGTLVTVSS 2640GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSTLSINAMGWFRQAPGKEREGVSWISSSDGSTYYAD76 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGRWGQGTLVTVSS 2641GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSSVSIDAMGWFRQAPGKEREFVAGISRSGDTTYYAD77 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNYYYPISRDEYDWGQGTL VTVSS2642 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKERELVAVTWSGGSTNYAD 78SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTLVTVSS 2643GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAVVNWSGRRTYYA79 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASSRMGVDDPETYGWGQGTLVTVSS 2644GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVAAVRWRGGITWY80 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDGLPIKYDWGQGTLV TVSS2645 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWFRQAPGKERELVASISRFGRTNYADSV 81KGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANGIESWGQGTLVTVSS 2646 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREFVASITSGGRMWYA 82DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTLVTVSS 2647 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFRFSSYGMGWFRQAPGKEREGVAAIGSDGSTSYADS 83VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQVWGQGTLVTVSS 2648 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFDNYNMGWFRQAPGKEREFVAAISWNGVTIYYA 84DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDWGQGTLVTVSS 2649 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYSMGWFRQAPGKEREFVAAISSGGLKAYADS 85VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDDYSGSWGQGTLVTVSS 2650 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGYTFRAYVMGWFRQAPGKERELLAVITRDGSTHYAD 86SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNGRWRSWSSRNPWGQGTLVTVSS 2651GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAAISRGSNSTDYAD87 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYTDYDLWGQGTLVTVSS 2652GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTISSYAMGWFRQAPGKERELVAAISKSSISTYYADS 88VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALGPVRRSRLEWGQGTLVTVSS 2653 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISWTGDSSSDG 89DTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAIFDVTDYERADWGQGTLV TVSS 2654GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTLGNYAMGWFRQAPGKERELVSAITWSDGSSYYA90 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASTRFAGRWGQGTLVTVSS 2655 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGNIDRLYAMGWFRQAPGKEREPVAAISPAAVTAGMT 91YYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSGSYYYTDDELDWGQGTL VTVSS 2656GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFGRRAMGWFRQAPGKERELVAAIRWSGKETWY92 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGNGGRTYGHSRARYEWGQGTLV TVSS2657 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFSIGAMGWFRQAPGKEREYVGSITWRGGNTYYA 93DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGVTGGAAYGWGQGTLVTVSS 2658 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYWMGWFRQAPGKEREVVAVIYTSDGSTYYA 94DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATIDGSWREWGQGTLVTVSS 2659 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGFGIDfyAMGWFRQAPGKEREFVAAISGSGDDTYYAD 95SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLELFHDEYNWGQGTLVT VSS 2660GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNILSLNTMGWFRQAPGKEREFVASVTWGFGSTSYAD96 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTLVTVSS 2661GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIYSLDAMGWFRQAPGKEREFVAAISPAALTTYYAD97 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAGSSRIYIYSDSLSERSYDWGQGTLVTVS S2662 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYGMGWFRQAPGKERELVAIKFSGGTTDYADS 98VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTLVTVSS 2663 GLP1R-41-EVQLVESGGGLVQPGGSLRLSCAASGRTFSKYAMGWFRQAPGKEREFVAAIRWSGGTTFYA 99DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGGWGTGRYNWGQGTLVTVSS 2664 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSIFSIYAMDWFRQAPGKEREFVAAISSDDSTTYYADS 01VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTAVLPAYDDWGQGTLVTVSS 2665 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSYISSSDGRTYYAD 02SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGLNGAAAAWGQGTLVTVSS 2666 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFSNGPMGWFRQAPGKEREFVAHISTGGATNYADS 03VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQGWGQGTLVTVSS 2667 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRALSSYSMGWFRQAPGKEREFVALITRSGGTTFYAD 04SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRHSYVDWGQGTLVTVSS 2668 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSIGSINAMGWFRQAPGKEREFVAAISWSGGATNYAD 05SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASVAYSDYDLGNDWGQGTLVTVSS 2669GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGLSFDDYAMGWFRQAPGKEREFVAAISGRSGNTYYA06 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALIQRRAPYSRLETWGQGTLVTVSS 2670GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMGWFRQAPGKEREGVAAISWSGGTTYYAD07 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAAGWVAEYGYWGQGTLVTVSS 2671GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVATISSNGNTTYYAD08 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLRVLRLRRYEYNYWGQGTLVTVSS 2672GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFRSNAMGWFRQAPGKEREGVAAISTSGGITYYAD09 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAERDGYGYWGQGTLVTVSS 2673 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKERELVAGISWNGGITYYA 10DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVRAGYDYWGQGTLVTVSS 2674 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREWVATISWSGGSTNYA 11DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVGRSGRDYWGQGTLVTVSS 2675 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRAFESYAMGWFRQAPGKEREFVAAIRWSGGSTYYA 12DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATGGWGTGRYNWGQGTLVTVSS 2676 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRIFSDYAMGWFRQAPGKEREFVATINGDGDSTNYAD 13SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANTYWYYTYDSWGQGTLVTVSS 2677 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRIFSDYAMGWFRQAPGKEREFVATINGDGDSTNYAD 14SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANTYCNYTYDSWGQGTLVTVSS 2678 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTLSRSNMGWFRQAPGKEREFVAAVRWSGGITWYA 15DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALGPVRRSRLEWGQGTLVTVSS 2679 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMGWFRQAPGKEREFVAAITWSGGSTNYA 16DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRAGRDSWGQGTLVTVSS 2680 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFNSYAMGWFRQAPGKEREFVAGITRSAVSTSYAD 17SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTLVTVSS 2681 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYVMGWFRQAPGKEREFVASITWSGGTTYYA 18DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRGSGRDYWGQGTLVTVSS 2682 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRALSSNSMGWFRQAPGKEREFVALITRSGGTTFYAD 19SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALNNRRRYVDWGQGTLVTVSS 2683 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 20DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVGRNGRDYWGQGTLVTVSS 2684 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISWSGGNTYYAD 21SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVPTIAYNTGYDYWGQGTLVTVSS 2685GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRIFDDYAMGWFRQAPGKERELVSGITWSGGSTYYA22 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGYDGYDYWGQGTLVTVSS 2686GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKERELVSAISTDDGSTYYAD23 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALPDDTYLATTYDYWGQGTLVTVSS 2687GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSIFSDNVMGWFRQAPGKEREMVAAIRWSGGITWYA24 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGRGDVSEYEYDWGQGTLVTVS S 2688GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGEIASIIAMGWFRQAPGKEREWVSAINSGGDTYYADS25 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRSRTIWPDWGQGTLVTVSS 2689GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSVSTMGWFRQAPGKEREIVAAITWSGSATYYAD26 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQRRWSQDWGQGTLVTVSS 2690 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKERELVAGITGGGSSTYYAD 27SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTRYGYDYWGQGTLVTVSS 2691 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGIPFRSRTMGWFRQAPGKEREFVAGITRNSIRTRYADS 28VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAPRRPYLPIRIRDYIWGQGTLVTVSS 2692GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTIVPYTMGWFRQAPGKEREFVAAISWSGASTIYAD29 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIGGTLYDRRRFEWGQGTLVTVSS 2693GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSNNAMGWFRQAPGKEREGVAAINGSGSITYYAD30 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAARDDYGYWGQGTLVTVSS 2694 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYGMGWFRQAPGKEREGVAGISWSDGSTSYAD 31SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASDASFDYWGQGTLVTVSS 2695 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGGTFSDYGMGWFRQAPGKEREGVASISWNDGSTSYA 32DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATADYDYWGQGTLVTVSS 2696 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSTFSTYAMGWFRQAPGKERELVAAISWSSGTTYYAD 33SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLVTSDGVSEYNYWGQGTLVTVSS 2697GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFLFDSYAMGWFRQAPGKEREPVAAISPAALTTYYAD34 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGTLVT VSS2698 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYAMGWFRQAPGKEREGVAAISWNSGSTYYA 35DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDARRYGYWGQGTLVTVSS 2699 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSTFGNYAMGWFRQAPGKEREFVAAISRSGSITYYAD 36SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDEDYALGPNEYDWGQGTLVTVSS 2700GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKERELVAGISWGGDSTYYA37 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGNGYDYWGQGTLVTVSS 2701GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSYISSSDGRTYYAD38 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTLVTVSS 2702GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGLTFWTSGMGWFRQAPGKEREYVAAISRSGSLKGYA39 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATVATALIWGQGTLVTVSS 2703 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTFSINAMGWFRQAPGKERELVSGISWGGGSTYYAD 40SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVNEDGFDYWGQGTLVTVSS 2704 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTFDDNAMGWFRQAPGKERELVAAISTSGSNTYYA 41DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAELREYGYWGQGTLVTVSS 2705 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFTSYNMGWFRQAPGKEREFLGSILWSDDSTNYAD 42SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQVWGQGTLVTVSS 2706 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYVMGWFRQAPGKEREFVAAINWNGSITYYA 43DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRSARNYWGQGTLVTVSS 2707 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISTSGGITYYAD 44SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDRIEYSRGGYDYWGQGTLVTVSS 2708GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFRKYAMGWFRQAPGKEREFVAAISSGGGSTNYA45 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRYRERDSWGQGTLVTVSS 2709GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISWSGDTTYYAD46 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIDLPDDTYLATEYDYWGQGTLVTVSS 2710GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSGFSPNVMGWFRQAPGKERELVAIKFSGGIIDYADS47 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTLVTVSS 2711GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTLTNHDMGWFRQAPGKEREGVSYISMSDGRTYYA48 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTLVTVSS 2712GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISRSGDSTYYAD49 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTLDNYGYWGQGTLVTVSS 2713GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTASSYHMGWFRQAPGKEREFVAFIHRSGTSTYYAD50 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADSITDRRSVAVAHTSYYWGQGTLVT VSS2714 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYAMGWFRQAPGKEREIVAAITWSGGITYYAD 51SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHGSILLDRIEWGQGTLVTVSS 2715 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGGTFSIYAMGWFRQAPGKERELVAAISSSGSITYYADS 52VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAAALDGPGDMYDYWGQGTLVTVSS 2716GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFDNYAMGWFRQAPGKERELVSGINSDGGSTYYA53 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVPISSPSDRNYWGQGTLVTVSS 2717GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSLTAMGWFRQAPGKEREFVAAISPAALTTYYAD54 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASRRAFRLSSDYEWGQGTLVTVSS 2718GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVAAVNWNGDSTYY55 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANWKMLLGVENDWGQGTLVTVSS 2719GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMGWFRQAPGKERELVAGISSSGGSTYYAD56 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGTYDYWGQGTLVTVSS 2720 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTFDIYAMGWFRQAPGKERELVAAINRDDSSTYYAD 57SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGLGNYNYWGQGTLVTVSS 2721 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRSFSFNAMGWFRQAPGKERELVAAITKLGFRNYADS 58VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASIEGVSGRWGQGTLVTVSS 2722 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSFFSINAMGWFRQAPGKERELVSASTWNGGYTYYA 59DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTLVTV SS 2723GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYAMGWFRQAPGKEREFVAGITSSGGYTYYA60 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVYYGDWEGSEPVQHEYDWGQGT LVTVSS2724 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSIFSRNAMGWFRQAPGKEREFVAAIRWSGKETWYA 61DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDWGQGTLVTVS S 2725GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFDTYAMGWFRQAPGKEREFVAGISGDGTITYYAD62 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDNPYWSGYNYWGQGTLVTVSS 2726GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFSNYAMGWFRQAPGKERELVSGINSDGGSTYYA63 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVSTNDGYDYWGQGTLVTVSS 2727GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGIYRVNTMGWFRQAPGKERELVAIKFSGGTTDYADS64 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTLVTVSS 2728GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMGWFRQAPGKERELVAGISSSGSSTYYAD65 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVSDGGYDYWGQGTLVTVSS 2729GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTSSIYNMGWFRQAPGKEREFVAAISRSGRSTSYADS 66VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTLVTVSS 2730GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRALSSYSMGWFRQAPGKEREFVALITRSGGTTFYAD67 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDWGQGTLVTVSS 2731GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRALSRYGMVWFRQAPGKEREFVAAINRGGKISHYA68 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGNGGRNYGHSRARYEWGQGTLVT VSS 2732GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFKFNDSYMRWFRQAPGKEREFVVAINWSSGSTYYA69 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVNGPIFWGQGTLVTVSS 2733 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRTLSDYALGWFRQAPGKERELVSGINTSGDTTYYAD 70SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVTSSYDYWGQGTLVTVSS 2734 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGSTFDIYGMGWFRQAPGKEREGVAAITGDGSSTSYAD 71SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADNDTEYGYWGQGTLVTVSS 2735 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGGTLDIYAMGWFRQAPGKEREFVAAISWSGSTTYYA 72DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGYDRDYWGQGTLVTVSS 2736 GLP1R-44-EVQLVESGGGLVQPGGSLRLSCAASGRPYSYDAMGWFRQAPGKEREIVAAISRTGSSIYYAD 73SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDGLPIKYDWGQGTLVTV SS 2737GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFRTYGMGWFRQAPGKEREGVAAISWSGNSTSYA74 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARLSKRGNRSSRDYWGQGTLVTVSS 2738GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFDNYAMGWFRQAPGKERELVAGINWSDSSTYYA75 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGWGEYDYWGQGTLVTVSS 2739GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKERELVAGINWSDSSTYYAD76 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTDYDEYNYWGQGTLVTVSS

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. A nucleic acid library, comprising: a plurality of nucleic acids,wherein each of the nucleic acids encodes for a sequence that whentranslated encodes for a GLP1R binding immunoglobulin, wherein the GLP1Rbinding immunoglobulin comprises a variant of a GLP1R binding domain,wherein the GLP1R binding domain is a ligand for the GLP1R, and whereinthe nucleic acid library comprises at least 10,000 variantimmunoglobulin heavy chains and at least 10,000 variant immunoglobulinlight chains.
 2. The nucleic acid library of claim 1, wherein thenucleic acid library comprises at least 50,000 variant immunoglobulinheavy chains and at least 50,000 variant immunoglobulin light chains. 3.(canceled)
 4. The nucleic acid library of claim 1, wherein the nucleicacid library comprises at least 10⁵ non-identical nucleic acids.
 5. Thenucleic acid library of claim 1, wherein a length of the immunoglobulinheavy chain when translated is about 90 to about 100 amino acids.
 6. Thenucleic acid library of claim 1, wherein a length of the immunoglobulinheavy chain when translated is about 100 to about 400 amino acids. 7.The nucleic acid library of claim 1, wherein the variant immunoglobulinheavy chain when translated comprises at least 90% sequence identity toSEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319,2320, or
 2321. 8. The nucleic acid library of claim 1, wherein thevariant immunoglobulin light chain when translated comprises at least90% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315,or
 2316. 9. A nucleic acid library comprising: a plurality of nucleicacids, wherein each of the nucleic acids encodes for a sequence thatwhen translated encodes for a GLP1R single domain antibody, wherein eachsequence of the plurality of sequences comprises a variant sequenceencoding for at least one of a CDR1, CDR2, and CDR3 on a heavy chain;wherein the library comprises at least 30,000 variant sequences; andwherein the antibody or antibody fragments bind to its antigen with aK_(D) of less than 100 nM.
 10. (canceled)
 11. (canceled)
 12. The nucleicacid library of claim 9, wherein the nucleic acid library comprises atleast 10⁵ non-identical nucleic acids.
 13. The nucleic acid library ofclaim 9, wherein a length of the heavy chain when translated is about 90to about 100 amino acids.
 14. The nucleic acid library of claim 9,wherein a length of the heavy chain when translated is about 100 toabout 400 amino acids.
 15. The nucleic acid library of claim 9, whereinthe heavy chain when translated comprises at least 90% sequence identityto SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318,2319, 2320, or
 2321. 16. (canceled)
 17. An antibody or antibody fragmentthat binds GLP1R, comprising an immunoglobulin heavy chain and animmunoglobulin light chain: a. wherein the immunoglobulin heavy chaincomprises an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317,2318, 2319, 2320, or 2321; and b. wherein the immunoglobulin light chaincomprises an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316.18.-24. (canceled)
 25. The antibody or antibody fragment of claim 17,wherein the antibody is a monoclonal antibody, a polyclonal antibody, abi-specific antibody, a multispecific antibody, a grafted antibody, ahuman antibody, a humanized antibody, a synthetic antibody, a chimericantibody, a camelized antibody, a single-chain Fvs (scFv), a singlechain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fvfragment, a single-domain antibody, an isolated complementaritydetermining region (CDR), a diabody, a fragment comprised of only asingle monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof.
 26. (canceled)
 27. The antibody or antibody fragmentof claim 17, wherein the antibody has an EC50 less than about 25nanomolar in a cAMP assay. 28.-33. (canceled)
 34. The antibody orantibody fragment of claim 17, wherein the antibody or antibody fragmentcomprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282,2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297,2298, 2299, 2300, 2301, or 2302 or a sequence set forth in Table
 27. 35.(canceled)
 36. An antagonist of GLP1R comprising SEQ ID NO: 2279 or2320.
 37. The antagonist of claim 36, wherein the antagonist comprisesan EC50 of no more than 1.5 nM.
 38. (canceled)
 39. (canceled) 40.(canceled)
 41. An agonist of GLP1R comprising SEQ ID NO:
 2317. 42. Theagonist of claim 41, wherein the agonist comprises an EC50 of no morethan 1.5 nM. 43.-57. (canceled)